Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

• MeSH
• kalibrace MeSH
• reprodukovatelnost výsledků MeSH
• ultracentrifugace metody   normy   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

A novel and rapid sample pretreatment technique based on a combination of ultracentrifugation and solid-phase extraction for the determination of α-tocopherol in human erythrocyte membranes by high-performance liquid chromatography with ultraviolet detection is presented in this work. Red blood cell samples were ultracentrifuged (288 000 × g, 3 min, 4°C) in the presence of d-mannitol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and calcium chloride. The α-tocopherol was then extracted from the erythrocyte membranes by solid-phase extraction with n-hexane in the presence of ascorbic acid. Tocopherol acetate was used as the internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP-18e (100 × 4.6 mm) using 100% methanol as the mobile phase. The absorbance of α-tocopherol was measured at a wavelength of 295 nm. The method was validated and showed sufficient accuracy and precision, ranging from 96.4 to 100.8% and from 4.5 to 6.3%, respectively. Moreover, the developed method was applied to the determination of erythrocyte α-tocopherol in real samples from patients. The combined ultracentrifugation and solid-phase extraction technique substantially decreased the time for the sample pretreatment step compared to liquid-liquid extraction and could be applicable for the quantitation of other analytes in erythrocyte membranes.

• Klíčová slova
• Alpha-tocopherol, Erythrocyte membranes, Red blood cells, Solid-phase extraction, Ultracentrifugation,
• Publikační typ
• časopisecké články MeSH

• MeSH
• arterioskleróza krev   MeSH
• chemické techniky analytické MeSH
• elektroforéza sérových bílkovin MeSH
• lidé MeSH
• lipoproteiny krev   MeSH
• metody MeSH
• ultracentrifugace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• lipoproteiny MeSH

• MeSH
• antigeny * MeSH
• antisérum MeSH
• chemické techniky analytické MeSH
• drůbež MeSH
• hemaglutinační testy MeSH
• imunoelektroforéza MeSH
• imunologická tolerance * MeSH
• kinetika MeSH
• merkaptoethanol farmakologie   MeSH
• precipitinové testy MeSH
• reakce antigenu s protilátkou MeSH
• sérový albumin * MeSH
• techniky in vitro MeSH
• tvorba protilátek * MeSH
• ultracentrifugace MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny * MeSH
• antisérum MeSH
• merkaptoethanol MeSH
• sérový albumin * MeSH

Contactin-associated protein-like 2 (CNTNAP2) encodes for CASPR2, a multidomain single transmembrane protein belonging to the neurexin superfamily that has been implicated in a broad range of human phenotypes including autism and language impairment. Using a combination of biophysical techniques, including small angle x-ray scattering, single particle electron microscopy, analytical ultracentrifugation, and bio-layer interferometry, we present novel structural and functional data that relate the architecture of the extracellular domain of CASPR2 to a previously unknown ligand, Contactin1 (CNTN1). Structurally, CASPR2 is highly glycosylated and has an overall compact architecture. Functionally, we show that CASPR2 associates with micromolar affinity with CNTN1 but, under the same conditions, it does not interact with any of the other members of the contactin family. Moreover, by using dissociated hippocampal neurons we show that microbeads loaded with CASPR2, but not with a deletion mutant, co-localize with transfected CNTN1, suggesting that CNTN1 is an endogenous ligand for CASPR2. These data provide novel insights into the structure and function of CASPR2, suggesting a complex role of CASPR2 in the nervous system.

• Klíčová slova
• analytical ultracentrifugation, ligand-binding protein, molecular cell biology, protein structure, small-angle x-ray scattering (SAXS),
• MeSH
• difrakce rentgenového záření MeSH
• HEK293 buňky MeSH
• hipokampus cytologie   metabolismus   MeSH
• kontaktin 1 metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• maloúhlový rozptyl MeSH
• mapy interakcí proteinů MeSH
• membránové proteiny chemie   metabolismus   ultrastruktura   MeSH
• molekulární modely MeSH
• myši inbrední C57BL MeSH
• proteiny nervové tkáně chemie   metabolismus   ultrastruktura   MeSH
• terciární struktura proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• CNTN1 protein, human MeSH Prohlížeč
• CNTNAP2 protein, human MeSH Prohlížeč
• kontaktin 1 MeSH
• membránové proteiny MeSH
• proteiny nervové tkáně MeSH

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) but the precise mechanism of this regulation is unclear due to the lack of structural data. Here, we performed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1. ASK1-TBD is a monomeric and rigid domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. The binding interaction does not involve the formation of intermolecular disulfide bonds. Residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp(31) being directly involved in the binding interaction as suggested by time-resolved fluorescence. Small-angle x-ray scattering data reveal a compact and slightly asymmetric shape of ASK1-TBD and suggest reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change.

• Klíčová slova
• Analytical Ultracentrifugation, Apoptosis Signal-regulating Kinase 1 (ASK1), Circular Dichroism (CD), Fluorescence, Small-angle X-ray Scattering (SAXS), Thioredoxin,
• MeSH
• biofyzika MeSH
• cirkulární dichroismus MeSH
• fluorescenční spektrometrie MeSH
• konformace proteinů MeSH
• MAP kinasa-kinasa-kinasa 5 metabolismus   MeSH
• oxidace-redukce MeSH
• thioredoxiny metabolismus   MeSH
• ultracentrifugace MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• MAP kinasa-kinasa-kinasa 5 MeSH
• thioredoxiny MeSH

BACKGROUND: In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS: By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS: There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. CONCLUSIONS: The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.

• MeSH
• Caenorhabditis elegans chemie   MeSH
• chemická frakcionace metody   MeSH
• cirkulující mikroRNA krev   izolace a purifikace   MeSH
• extracelulární vezikuly chemie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové biomarkery krev   izolace a purifikace   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• senioři MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• zvířata MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cirkulující mikroRNA MeSH
• nádorové biomarkery MeSH

The opportunistic Gram-negative bacterium Burkholderia cenocepacia causes lethal infections in cystic fibrosis patients. Multivalent mannoside derivatives were prepared as potential inhibitors of lectin BC2L-A, one of the virulence factors deployed by B. cenocepacia in the infection process. An (α1→2)-thio-linked mannobioside mimic bearing an azide functionalized aglycon was conjugated to different multivalent scaffolds such as propargylated calix[4]arenes, methyl gallate and pentaerythritol by azide-alkyne 1,3-dipolar cycloaddition. The interaction between the glycoclusters and the mannose binding BC2L-A lectin from B. cenocepacia was examined by isothermal microcalorimetry, surface plasmon resonance, inhibition of yeast agglutination and analytical ultracentrifugation.

• Klíčová slova
• Anti-adhesion therapy, Burkholderia cenocepacia, Click reaction, Glycoclusters, Mannose-binding lectin,
• MeSH
• aglutinační testy MeSH
• Burkholderia cenocepacia chemie   MeSH
• kalorimetrie metody   MeSH
• kvasinky účinky léků   MeSH
• lektin vázající mannosu chemie   metabolismus   farmakologie   MeSH
• ligandy MeSH
• mannosidy chemická syntéza   chemie   metabolismus   MeSH
• povrchová plasmonová rezonance MeSH
• techniky syntetické chemie MeSH
• ultracentrifugace metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• lektin vázající mannosu MeSH
• ligandy MeSH
• mannosidy MeSH

Human histone deacetylase 6 (HDAC6) is a structurally unique, multidomain protein implicated in a variety of physiological processes including cytoskeletal remodelling and the maintenance of cellular homeostasis. Our current understanding of the HDAC6 structure is limited to isolated domains, and a holistic picture of the full-length protein structure, including possible domain interactions, is missing. Here, we used an integrative structural biology approach to build a solution model of HDAC6 by combining experimental data from several orthogonal biophysical techniques complemented by molecular modelling. We show that HDAC6 is best described as a mosaic of folded and intrinsically disordered domains that in-solution adopts an ensemble of conformations without any stable interactions between structured domains. Furthermore, HDAC6 forms dimers/higher oligomers in a concentration-dependent manner, and its oligomerization is mediated via the positively charged N-terminal microtubule-binding domain. Our findings provide the first insights into the structure of full-length human HDAC6 and can be used as a basis for further research into structure function and physiological studies of this unique deacetylase.

• Klíčová slova
• acetylation, analytical ultracentrifugation, intrinsically disordered regions, oligomerization, small-angle X-ray scattering,
• MeSH
• acetylace MeSH
• histondeacetylasa 6 genetika   chemie   metabolismus   MeSH
• histondeacetylasy * metabolismus   MeSH
• inhibitory histondeacetylas MeSH
• lidé MeSH
• mikrotubuly * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histondeacetylasa 6 MeSH
• histondeacetylasy * MeSH
• inhibitory histondeacetylas MeSH

Protein-protein interactions (PPIs) remain poorly explored targets for the treatment of Alzheimer's disease. The interaction of 14-3-3 proteins with Tau was shown to be linked to Tau pathology. This PPI is therefore seen as a potential target for Alzheimer's disease. When Tau is phosphorylated by PKA (Tau-PKA), several phosphorylation sites are generated, including two known 14-3-3 binding sites, surrounding the phosphorylated serines 214 and 324 of Tau. The crystal structures of 14-3-3 in complex with peptides surrounding these Tau phosphosites show that both these motifs are anchored in the amphipathic binding groove of 14-3-3. However, in the absence of structural data with the full-length Tau protein, the stoichiometry of the complex or the interface and affinity of the partners is still unclear. In this work, we addressed these points, using a broad range of biophysical techniques. The interaction of the long and disordered Tau-PKA protein with 14-3-3σ is restricted to two short sequences, containing phosphorylated serines, which bind in the amphipathic binding groove of 14-3-3σ. Phosphorylation of Tau is fundamental for the formation of this stable complex, and the affinity of the Tau-PKA/14-3-3σ interaction is in the 1-10 micromolar range. Each monomer of the 14-3-3σ dimer binds one of two different phosphorylated peptides of Tau-PKA, suggesting a 14-3-3/Tau-PKA stoichiometry of 2 : 1, confirmed by analytical ultracentrifugation. These results contribute to a better understanding of this PPI and provide useful insights for drug discovery projects aiming at the modulation of this interaction.

• Klíčová slova
• 14-3-3 proteins, Alzheimer’s disease, NMR spectroscopy, Tau protein, analytical ultracentrifugation, protein-protein interactions,
• MeSH
• Alzheimerova nemoc genetika   metabolismus   patologie   MeSH
• exoribonukleasy chemie   genetika   metabolismus   MeSH
• fosforylace MeSH
• konformace proteinů MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• multimerizace proteinu * MeSH
• mutace MeSH
• povrchová plasmonová rezonance MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• proteiny 14-3-3 chemie   genetika   metabolismus   MeSH
• proteiny tau chemie   genetika   metabolismus   MeSH
• serin chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• exoribonukleasy MeSH
• proteinkinasy závislé na cyklickém AMP MeSH
• proteiny 14-3-3 MeSH
• proteiny tau MeSH
• serin MeSH
• SFN protein, human MeSH Prohlížeč