• MeSH
• nukleární lékařství MeSH
• Publikační typ
• periodika MeSH
• příležitostné publikace MeSH

• Konspekt
• Lékařské vědy. Lékařství
• NLK Obory
• radiologie, nukleární medicína a zobrazovací metody

Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for tracking NPM aggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population of NPM labeled either with eGFP or mRFP1. We observe joint oligomers formed from the fluorescently labeled NPM. Having validated the in vivo methods, we study an effect of substitutions in cysteine 21 (Cys21) of the NPM N-terminus on the oligomerization to demonstrate applicability of the methods. Inhibitory effect of mutations of the Cys21 to nonpolar Ala or to aromatic Phe on the oligomerization was reported in literature using in vitro semi-native electrophoresis. However, we do not detect any break-up of the joint NPM oligomers due to the Cys21 mutations in live cells. In vivo microscopy observations are supported by an in vitro method, the GFP-Trap immunoprecipitation assay. Our results therefore show importance of utilizing several methods for detection of biologically relevant protein aggregates. In vivo monitoring of the NPM oligomerization, a potential cancer therapy target, by the presented methods offers a new way to monitor effects of drugs that are tested as NPM oligomerization inhibitors directly in live cells.

• MeSH
• jaderné proteiny metabolismus   MeSH
• lidé MeSH
• proteinové agregáty fyziologie   MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Chromatin architect of muscle expression (Charme) is a muscle-restricted long noncoding RNA (lncRNA) that plays an important role in myogenesis. Earlier evidence indicates that the nuclear Charme isoform, named pCharme, acts on the chromatin by assisting the formation of chromatin domains where myogenic transcription occurs. By combining RNA antisense purification (RAP) with mass spectrometry and loss-of-function analyses, we have now identified the proteins that assist these chromatin activities. These proteins-which include a sub-set of splicing regulators, principally PTBP1 and the multifunctional RNA/DNA binding protein MATR3-bind to sequences located within the alternatively spliced intron-1 to form nuclear aggregates. Consistent with the functional importance of pCharme interactome in vivo, a targeted deletion of the intron-1 by a CRISPR-Cas9 approach in mouse causes the release of pCharme from the chromatin and results in cardiac defects similar to what was observed upon knockout of the full-length transcript.

• MeSH
• heterogenní jaderné ribonukleoproteiny metabolismus   MeSH
• introny genetika   MeSH
• lidé MeSH
• myši MeSH
• protein vázající polypyrimidinové úseky RNA metabolismus   MeSH
• proteiny asociované s jadernou matrix metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• RNA dlouhá nekódující metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Intracellular protein aggregation causes proteotoxic stress, underlying highly debilitating neurodegenerative disorders in parallel with decreased proteasome activity. Nevertheless, under such stress conditions, the expression of proteasome subunits is upregulated by Nuclear Factor Erythroid 2-related factor 1 (NRF1), a transcription factor that is encoded by NFE2L1. Activating the NRF1 pathway could accordingly delay the onset of neurodegenerative and other disorders with impaired cell proteostasis. Here, we present a series of small-molecule compounds based on bis(phenylmethylen)cycloalkanones and their heterocyclic analogues, identified via targeted library screening, that can induce NRF1-dependent downstream events, such as proteasome synthesis, heat shock response, and autophagy, in both model cell lines and Caenorhabditis elegans strains. These compounds increase proteasome activity and decrease the size and number of protein aggregates without causing any cellular stress or inhibiting the ubiquitin-proteasome system (UPS). Therefore, our compounds represent a new promising therapeutic approach for various protein conformational diseases, including the most debilitating neurodegenerative diseases.

• MeSH
• aktivace transkripce účinky léků   MeSH
• autofagie účinky léků   MeSH
• Caenorhabditis elegans * účinky léků   metabolismus   MeSH
• faktor 1 související s NF-E2 metabolismus   genetika   MeSH
• knihovny malých molekul farmakologie   MeSH
• lidé MeSH
• patologická konformace proteinů metabolismus   farmakoterapie   MeSH
• proteasomový endopeptidasový komplex * metabolismus   MeSH
• proteinové agregáty * účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Spinocerebellar ataxia type-1 (SCA1) is caused by an abnormally expanded polyglutamine (polyQ) tract in ataxin-1. These expansions are responsible for protein misfolding and self-assembly into intranuclear inclusion bodies (IIBs) that are somehow linked to neuronal death. However, owing to lack of a suitable cellular model, the downstream consequences of IIB formation are yet to be resolved. Here, we describe a nuclear protein aggregation model of pathogenic human ataxin-1 and characterize IIB effects. Using an inducible Sleeping Beauty transposon system, we overexpressed the ATXN1(Q82) gene in human mesenchymal stem cells that are resistant to the early cytotoxic effects caused by the expression of the mutant protein. We characterized the structure and the protein composition of insoluble polyQ IIBs which gradually occupy the nuclei and are responsible for the generation of reactive oxygen species. In response to their formation, our transcriptome analysis reveals a cerebellum-specific perturbed protein interaction network, primarily affecting protein synthesis. We propose that insoluble polyQ IIBs cause oxidative and nucleolar stress and affect the assembly of the ribosome by capturing or down-regulating essential components. The inducible cell system can be utilized to decipher the cellular consequences of polyQ protein aggregation. Our strategy provides a broadly applicable methodology for studying polyQ diseases.

• MeSH
• ataxin-1 genetika   metabolismus   MeSH
• intranukleární inkluzní tělíska * metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• oxidační stres MeSH
• proteiny nervové tkáně * genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• blastomery fyziologie   MeSH
• chiméra MeSH
• fertilizace in vitro metody   MeSH
• finanční podpora výzkumu jako téma MeSH
• oocyty cytologie   fyziologie   MeSH
• přenos embrya MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH

Galectins have the particular capacity to interact with distinct proteins, in addition to the typical reactivity of lectins with glycans. Therefore, they can be functionally active when residing at places other than the membrane or extracellular matrix. In fact, nuclear presence of galectins-1 and -3 is solidly documented but it is an open question whether these two cases are exceptional within this lectin family. Thus, galectin-2, which shares 43% sequence identity on the protein level with galectin-1, warrants study in this respect. Based on initial immunohistochemical evidence we herein address the issue as to whether this galectin can join the category of nuclear lectins. To do so we studied different types of cell in vitro using an antibody preparation free of cross-reactivity against other tested galectins. The immunocytochemical experiments revealed that galectin-2 was present in nuclei of murine 3T3 fibroblasts and also genetically engineered human colon carcinoma cells with stable ectopic expression. Transport of galectin-2 to the nucleus could be enhanced by physical (UV light), chemical (mitomycin C, serum withdrawal) or cell biological (coculture with stromal cells) treatment modalities. As a means of further characterizing the staining profile cytochemically, a series of markers with well-defined site of residency within the nuclear compartment was tested in parallel. Importantly, no colocalization with galectins-1 and -3 and the splicing factor SC35 was detectable, the former cases also serving as inherent specificity control. In contrast, a similarity was uncovered in the case of the promyelocytic leukemia (PML) protein as marker of PML nuclear bodies. In aggregate, nuclear localization is documented for galectin-2. This attribute should thus not be considered as an exceptional finding confined to galectins-1 and -3. That even closely related family members, here galectins-1 and -2, exhibit distinct intranuclear localization patterns gives ensuing research a clear direction.

• MeSH
• adenokarcinom metabolismus   MeSH
• biologické markery MeSH
• biologický transport MeSH
• buněčné jádro metabolismus   MeSH
• buňky 3T3 MeSH
• financování organizované MeSH
• galektin 2 metabolismus   MeSH
• imunohistochemie MeSH
• kokultivační techniky MeSH
• kultivační média bez séra MeSH
• lidé MeSH
• mitomycin farmakologie   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory tračníku metabolismus   MeSH
• ultrafialové záření MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

Protein aggregates and abnormal proteins are toxic and associated with neurodegenerative diseases. There are several mechanisms to help cells get rid of aggregates but little is known on how cells prevent aggregate-prone proteins from being synthesised. The EBNA1 of the Epstein-Barr virus (EBV) evades the immune system by suppressing its own mRNA translation initiation in order to minimize the production of antigenic peptides for the major histocompatibility (MHC) class I pathway. Here we show that the emerging peptide of the disordered glycine-alanine repeat (GAr) within EBNA1 dislodges the nascent polypeptide-associated complex (NAC) from the ribosome. This results in the recruitment of nucleolin to the GAr-encoding mRNA and suppression of mRNA translation initiation in cis. Suppressing NAC alpha (NACA) expression prevents nucleolin from binding to the GAr mRNA and overcomes GAr-mediated translation inhibition. Taken together, these observations suggest that EBNA1 exploits a nascent protein quality control pathway to regulate its own rate of synthesis that is based on sensing the nascent GAr peptide by NAC followed by the recruitment of nucleolin to the GAr-encoding RNA sequence.

• MeSH
• alanin MeSH
• fosfoproteiny MeSH
• glycin MeSH
• infekce virem Epsteina-Barrové * MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• peptidy genetika   MeSH
• proteinové agregáty MeSH
• proteiny vázající RNA metabolismus   MeSH
• virus Epsteinův-Barrové - jaderné antigeny metabolismus   MeSH
• virus Epsteinův-Barrové * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The protein sequences found in nature represent a tiny fraction of the potential sequences that could be constructed from the 20-amino-acid alphabet. To help define the properties that shaped proteins to stand out from the space of possible alternatives, we conducted a systematic computational and experimental exploration of random (unevolved) sequences in comparison with biological proteins. In our study, combinations of secondary structure, disorder, and aggregation predictions are accompanied by experimental characterization of selected proteins. We found that the overall secondary structure and physicochemical properties of random and biological sequences are very similar. Moreover, random sequences can be well-tolerated by living cells. Contrary to early hypotheses about the toxicity of random and disordered proteins, we found that random sequences with high disorder have low aggregation propensity (unlike random sequences with high structural content) and were particularly well-tolerated. This direct structure content/aggregation propensity dependence differentiates random and biological proteins. Our study indicates that while random sequences can be both structured and disordered, the properties of the latter make them better suited as progenitors (in both in vivo and in vitro settings) for further evolution of complex, soluble, three-dimensional scaffolds that can perform specific biochemical tasks.

• MeSH
• cirkulární dichroismus MeSH
• databáze proteinů MeSH
• datové soubory jako téma MeSH
• molekulární modely * MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• peptidová knihovna * MeSH
• proteinové agregáty MeSH
• rekombinantní proteiny chemie   izolace a purifikace   toxicita   MeSH
• rozpustnost MeSH
• sbalování proteinů MeSH
• sekundární struktura proteinů * MeSH
• sekvence aminokyselin MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The coarse-grained Martini model is employed extensively to study membrane protein oligomerization. While this approach is exceptionally promising given its computational efficiency, it is alarming that a significant fraction of these studies demonstrate unrealistic protein clusters, whose formation is essentially an irreversible process. This suggests that the protein-protein interactions are exaggerated in the Martini model. If this held true, then it would limit the applicability of Martini to study multi-protein complexes, as the rapidly clustering proteins would not be able to properly sample the correct dimerization conformations. In this work we first demonstrate the excessive protein aggregation by comparing the dimerization free energies of helical transmembrane peptides obtained with the Martini model to those determined from FRET experiments. Second, we show that the predictions provided by the Martini model for the structures of transmembrane domain dimers are in poor agreement with the corresponding structures resolved using NMR. Next, we demonstrate that the first issue can be overcome by slightly scaling down the Martini protein-protein interactions in a manner, which does not interfere with the other Martini interaction parameters. By preventing excessive, irreversible, and non-selective aggregation of membrane proteins, this approach renders the consideration of lateral dynamics and protein-lipid interactions in crowded membranes by the Martini model more realistic. However, this adjusted model does not lead to an improvement in the predicted dimer structures. This implicates that the poor agreement between the Martini model and NMR structures cannot be cured by simply uniformly reducing the interactions between all protein beads. Instead, a careful amino-acid specific adjustment of the protein-protein interactions is likely required.

• MeSH
• chemické modely * MeSH
• dimerizace MeSH
• konformace proteinů MeSH
• mapy interakcí proteinů MeSH
• membránové proteiny chemie   metabolismus   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• rezonanční přenos fluorescenční energie MeSH
• termodynamika MeSH
• Publikační typ
• časopisecké články MeSH