• MeSH
• elektroforéza kapilární * metody   přístrojové vybavení   MeSH

Nuclear polyadenylated RNA-binding (Nab)3 protein is an RNA-binding protein that is involved in the poly(A) independent termination pathway. Here, we report the NMR spectral assignments of RNA-recognition motif (RRM) of Nab3. The assignment will allow performing NMR structural and RNA-binding studies of Nab3 with the aim to investigate its role in the poly(A) independent termination pathway.

• MeSH
• aminokyselinové motivy MeSH
• izotopy dusíku chemie   MeSH
• izotopy uhlíku chemie   MeSH
• jaderné proteiny chemie   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteiny vázající RNA chemie   MeSH
• Saccharomyces cerevisiae - proteiny chemie   MeSH
• Saccharomyces cerevisiae MeSH
• sekundární struktura proteinů MeSH
• vodík chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Fox-1 RNA recognition motif (RRM) domain is an important member of the RRM protein family. We report a 1.8 Å X-ray structure of the free Fox-1 containing six distinct monomers. We use this and the nuclear magnetic resonance (NMR) structure of the Fox-1 protein/RNA complex for molecular dynamics (MD) analyses of the structured hydration. The individual monomers of the X-ray structure show diverse hydration patterns, however, MD excellently reproduces the most occupied hydration sites. Simulations of the protein/RNA complex show hydration consistent with the isolated protein complemented by hydration sites specific to the protein/RNA interface. MD predicts intricate hydration sites with water-binding times extending up to hundreds of nanoseconds. We characterize two of them using NMR spectroscopy, RNA binding with switchSENSE and free-energy calculations of mutant proteins. Both hydration sites are experimentally confirmed and their abolishment reduces the binding free-energy. A quantitative agreement between theory and experiment is achieved for the S155A substitution but not for the S122A mutant. The S155 hydration site is evolutionarily conserved within the RRM domains. In conclusion, MD is an effective tool for predicting and interpreting the hydration patterns of protein/RNA complexes. Hydration is not easily detectable in NMR experiments but can affect stability of protein/RNA complexes.

• MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• motiv rozpoznávající RNA genetika   MeSH
• mutageneze cílená MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• RNA metabolismus   MeSH
• sestřihové faktory chemie   genetika   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• substituce aminokyselin MeSH
• vazebná místa MeSH
• voda chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

TDP-43 encodes an alternative-splicing regulator with tandem RNA-recognition motifs (RRMs). The protein regulates cystic fibrosis transmembrane regulator (CFTR) exon 9 splicing through binding to long UG-rich RNA sequences and is found in cytoplasmic inclusions of several neurodegenerative diseases. We solved the solution structure of the TDP-43 RRMs in complex with UG-rich RNA. Ten nucleotides are bound by both RRMs, and six are recognized sequence specifically. Among these, a central G interacts with both RRMs and stabilizes a new tandem RRM arrangement. Mutations that eliminate recognition of this key nucleotide or crucial inter-RRM interactions disrupt RNA binding and TDP-43-dependent splicing regulation. In contrast, point mutations that affect base-specific recognition in either RRM have weaker effects. Our findings reveal not only how TDP-43 recognizes UG repeats but also how RNA binding-dependent inter-RRM interactions are crucial for TDP-43 function.

• MeSH
• DNA vazebné proteiny chemie   metabolismus   fyziologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• protein CFTR genetika   metabolismus   MeSH
• proteiny vázající RNA chemie   metabolismus   fyziologie   MeSH
• sekvence aminokyselin MeSH
• sestřih RNA fyziologie   MeSH
• terciární struktura proteinů MeSH
• vazebná místa MeSH
• zastoupení bazí MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The RNA recognition motif (RRM) is the most common RNA binding domain across eukaryotic proteins. It is therefore of great value to engineer its specificity to target RNAs of arbitrary sequence. This was recently achieved for the RRM in Rbfox protein, where four mutations R118D, E147R, N151S, and E152T were designed to target the precursor to the oncogenic miRNA 21. Here, we used a variety of molecular dynamics-based approaches to predict specific interactions at the binding interface. Overall, we have run approximately 50 microseconds of enhanced sampling and plain molecular dynamics simulations on the engineered complex as well as on the wild-type Rbfox·pre-miRNA 20b from which the mutated systems were designed. Comparison with the available NMR data on the wild type molecules (protein, RNA, and their complex) served to establish the accuracy of the calculations. Free energy calculations suggest that further improvements in affinity and selectivity are achieved by the S151T replacement.

• MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• mikro RNA chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• motiv rozpoznávající RNA * genetika   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteinové inženýrství MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• RNA chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• stabilita RNA MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• výpočetní biologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Calix[4]arenes bearing urea units at the meta position(s) of the upper rim of the macrocyclic skeleton were prepared by the reaction of the corresponding amines with aryl isocyanates. As shown by the 1H NMR and UV/vis titration experiments, these systems are capable of effectively complexing selected anions even in a highly competitive environment (such as DMSO-d6). While the monoureido derivatives showed approximately the same complexation ability irrespective of the substitution (para vs. meta isomers), the bisureas at the upper rim demonstrated interesting differences in complexation. The meta,meta and para,para isomers were shown to prefer 2 : 1 complexes (anion : receptor) regardless of the anion tested, while the analogous meta,para isomer formed 1 : 1 complexes with strongly coordinated anions (e.g. H2PO4-) based on synchronous complexation by both ureido groups. This suggests that the regioselective introduction of urea units into the upper rim of calix[4]arene brings with it the possibility of "tuning" the complexation properties depending on the substitution pattern of the functional groups.

• Publikační typ
• časopisecké články MeSH

This paper describes the preparation and use of conjugates of porphyrins and bile acids as ligands to bind to tumor expressed saccharides. Bile acid-porphyrin conjugates were tested for recognition of saccharides that are typically present on malignant tumor cells. Fluorescence microscopy, in vitro PDT cell killing, and PDT of subcutaneous 4T1 mouse tumors is reported. High selectivity for saccharide cancer markers and cancer cells was observed. This in vivo and in vitro study demonstrated high potential use for these compounds in targeted photodynamic therapy.

• MeSH
• apoptóza účinky léků   MeSH
• buněčné linie MeSH
• buňky 3T3 MeSH
• financování organizované MeSH
• fluorescenční mikroskopie metody   MeSH
• fotochemoterapie metody   MeSH
• fragmentace DNA účinky léků   MeSH
• glykosylace MeSH
• HeLa buňky MeSH
• lidé MeSH
• ligandy MeSH
• molekulární struktura MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nádorové biomarkery analýza   MeSH
• nádory diagnóza   farmakoterapie   MeSH
• porfyriny farmakologie   chemie   MeSH
• proliferace buněk účinky léků   MeSH
• roztoky chemie   MeSH
• sacharidy chemie   MeSH
• screeningové testy protinádorových léčiv MeSH
• senzitivita a specificita MeSH
• transformované buněčné linie MeSH
• vazebná místa MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• žlučové kyseliny a soli farmakologie   chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

• MeSH
• cytosin chemie   MeSH
• finanční podpora výzkumu jako téma MeSH
• kvartérní amoniové sloučeniny farmakologie   chemie   MeSH
• metaloporfyriny chemie   MeSH
• molekulární struktura MeSH
• nukleotidy metabolismus   MeSH
• polyvinylchlorid chemie   MeSH
• techniky in vitro MeSH

The transient receptor potential ankyrin 1 channel (TRPA1) belongs to the TRP cation channel superfamily that responds to a panoply of stimuli such as changes in temperature, calcium levels, reactive oxygen and nitrogen species and lipid mediators among others. The TRP superfamily has been implicated in diverse pathological states including neurodegenerative disorders, kidney diseases, inflammation, pain and cancer. The intracellular C-terminus is an important regulator of TRP channel activity. Studies with this and other TRP superfamily members have shown that the C-terminus association with lipid bilayer alters channel sensitivity and activation, especially interactions occurring through basic residues. Nevertheless, it is not yet clear how this process takes place and which regions in the C-terminus would be responsible for such membrane recognition. With that in mind, herein the first putative membrane interacting region of the C-terminus of human TRPA1, (corresponding to a 29 residue peptide, IAEVQKHASLKRIAMQVELHTSLEKKLPL) named H1 due to its potential helical character was chosen for studies of membrane interaction. The affinity of H1 to lipid membranes, H1 structural changes occurring upon this interaction as well as effects of this interaction in lipid organization and integrity were investigated using a biophysical approach. Lipid models systems composed of zwitterionic and anionic lipids, namely those present in the lipid membrane inner leaflet, where H1 is prone to interact, where used. The study reveals a strong interaction and affinity of H1 as well as peptide structuration especially with membranes containing anionic lipids. Moreover, the interactions and peptide structure adoption are headgroup specific.

• MeSH
• anizotropie MeSH
• buněčná membrána chemie   metabolismus   MeSH
• kationtové kanály TRP chemie   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• membránové lipidy chemie   metabolismus   MeSH
• membrány umělé * MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• peptidové fragmenty chemie   metabolismus   MeSH
• proteiny nervové tkáně chemie   metabolismus   MeSH
• pufry MeSH
• sbalování proteinů MeSH
• sekundární struktura proteinů MeSH
• terciární struktura proteinů MeSH
• vápníkové kanály chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• změna skupenství MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• DNA MeSH
• extrachromozomální dědičnost MeSH
• molekulární struktura MeSH
• nukleové kyseliny MeSH
• osmium MeSH