Vestibular schwannoma is the most common benign neoplasm of the cerebellopontine angle. Its first symptoms include hearing loss, tinnitus, and vestibular symptoms, followed by cerebellar and brainstem symptoms, along with palsy of the adjacent cranial nerves. However, the clinical picture has unpredictable dynamics and currently, there are no reliable predictors of tumor behavior. Hence, it is desirable to have a fast routine method for analysis of vestibular schwannoma tissues at the molecular level. The major objective of this study was to verify whether a technique using in-sample specific protein digestion with trypsin would have the potential to provide a proteomic characterization of these pathological tissues. The achieved results showed that the use of this approach with subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of released peptides allowed a fast identification of a considerable number of proteins in two differential parts of vestibular schwannoma tissue as well as in tissues of control healthy samples. Furthermore, mathematical analysis of MS data was able to discriminate between pathological vestibular schwannoma tissues and healthy tissues. Thus, in-sample protein digestion combined with LC-MS/MS separation and identification of released specific peptides followed by mathematical analysis appears to have the potential for routine characterization of vestibular schwannomas at the molecular level. Data are available via ProteomeXchange with identifier PXD045261.
- MeSH
- chromatografie kapalinová metody MeSH
- lidé MeSH
- peptidové fragmenty * analýza chemie metabolismus MeSH
- peptidy metabolismus MeSH
- proteolýza MeSH
- proteomika metody MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- trypsin chemie MeSH
- vestibulární schwannom * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Atomic characterization of large nonfibrillar aggregates of amyloid polypeptides cannot be determined by experimental means. Starting from β-rich aggregates of Y and elongated topologies predicted by coarse-grained simulations and consisting of more than 100 Aβ16-22 peptides, we performed atomistic molecular dynamics (MD), replica exchange with solute scaling (REST2), and umbrella sampling simulations using the CHARMM36m force field in explicit solvent. Here, we explored the dynamics within 3 μs, the free energy landscape, and the potential of mean force associated with either the unbinding of one single peptide in different configurations within the aggregate or fragmentation events of a large number of peptides. Within the time scale of MD and REST2, we find that the aggregates experience slow global conformational plasticity, and remain essentially random coil though we observe slow beta-strand structuring with a dominance of antiparallel beta-sheets over parallel beta-sheets. Enhanced REST2 simulation is able to capture fragmentation events, and the free energy of fragmentation of a large block of peptides is found to be similar to the free energy associated with fibril depolymerization by one chain for longer Aβ sequences.
Preptin is a 34-amino-acid-long peptide derived from the E-domain of a precursor of insulin-like growth factor 2 (pro-IGF2) with bone-anabolic and insulin secretion amplifying properties. Here, we describe the synthesis, structures, and biological activities of six shortened analogues of human preptin. Eight- and nine-amino-acid-long peptide amides corresponding to the C-terminal part of human preptin were stabilised by two types of staples to induce a higher proportion of helicity in their secondary structure. We monitored the secondary structure of the stapled peptides using circular dichroism. The biological effect of the structural changes was determined afterwards by the ability of peptides to stimulate the release of intracellular calcium ions. We confirmed the previous observation that the stabilisation of the disordered conformation of human preptin has a deleterious effect on biological potency. However, surprisingly, one of our preptin analogues, a nonapeptide stabilised by olefin metathesis between positions 3 and 7 of the amino acid chain, had a similar ability to stimulate calcium ions' release to the full-length human preptin. Our findings could open up new ways to design new preptin analogues, which may have potential as drugs for the treatment of diabetes and osteoporosis.
- MeSH
- insulinu podobný růstový faktor II * chemie MeSH
- kosti a kostní tkáň MeSH
- lidé MeSH
- peptidové fragmenty chemie MeSH
- peptidy MeSH
- vápník * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85-111, 117-143, and 157-165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells via various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 in vivo. Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses.
- MeSH
- B-lymfocyty imunologie MeSH
- CD4-pozitivní T-lymfocyty imunologie MeSH
- CD8-pozitivní T-lymfocyty imunologie MeSH
- kopolymer kyseliny glykolové a mléčné chemie farmakologie MeSH
- lidé MeSH
- nádorové proteiny chemie farmakologie MeSH
- nanočástice chemie terapeutické užití MeSH
- nosiče léků chemie farmakologie MeSH
- peptidové fragmenty chemie farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In early stages of Alzheimer's disease (AD), amyloid beta (Aβ) accumulates in the mitochondrial matrix and interacts with mitochondrial proteins, such as cyclophilin D (cypD) and 17β-hydroxysteroid dehydrogenase 10 (17β-HSD10). Multiple processes associated with AD such as increased production or oligomerization of Aβ affect these interactions and disbalance the equilibrium between the biomolecules, which contributes to mitochondrial dysfunction. Here, we investigate the effect of the ionic environment on the interactions of Aβ (Aβ1-40, Aβ1-42) with cypD and 17β-HSD10 using a surface plasmon resonance (SPR) biosensor. We show that changes in concentrations of K+ and Mg2+ significantly affect the interactions and may increase the binding efficiency between the biomolecules by up to 35% and 65% for the interactions with Aβ1-40 and Aβ1-42, respectively, in comparison with the physiological state. We also demonstrate that while the binding of Aβ1-40 to cypD and 17β-HSD10 takes place preferentially around the physiological concentrations of ions, decreased concentrations of K+ and increased concentrations of Mg2+ promote the interaction of both mitochondrial proteins with Aβ1-42. These results suggest that the ionic environment represents an important factor that should be considered in the investigation of biomolecular interactions taking place in the mitochondrial matrix under physiological as well as AD-associated conditions.
- MeSH
- 17-hydroxysteroidní dehydrogenasy chemie genetika MeSH
- Alzheimerova nemoc diagnóza genetika patologie MeSH
- amyloidní beta-protein chemie MeSH
- biosenzitivní techniky metody MeSH
- ionty chemie MeSH
- lidé MeSH
- mitochondriální proteiny chemie MeSH
- mitochondrie chemie MeSH
- peptidové fragmenty chemie genetika MeSH
- peptidylprolylisomerasa F chemie genetika MeSH
- povrchová plasmonová rezonance metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Histone deacetylase 6 (HDAC6) is a multidomain cytosolic hydrolase acting mostly on nonhistone protein substrates. Investigations of the substrate specificity of HDAC6 are confounded by the presence of 2 catalytically active deacetylase domains (DD1 and DD2). In this study, acetylome peptide microarrays and peptide libraries were used to map the substrate specificity of DD1 and DD2 of human HDAC6. The results show that DD1 is solely responsible for the deacetylation of substrates harboring the acetyllysine at their C terminus, whereas DD2 exclusively deacetylates peptides with an internal acetyllysine residue. Also, statistical analysis of the deacetylation data revealed amino acid preferences at individual positions flanking the acetyllysine, where glycine and arginine residues are favored at positions N-terminal to the central acetyllysine; negatively charged glutamate is strongly disfavored throughout the sequence. Finally, the deacylation activity of HDAC6 was profiled by using a panel of acyl derivatives of the optimized peptide substrate and showed that HDAC6 acts as a proficient deformylase. Our data thus offer a detailed insight into the substrate preferences of the individual HDAC6 domains at the peptide level, and these findings can in turn help in elucidating the biologic roles of the enzyme and facilitate the development of new domain-specific inhibitors as research tools or therapeutic agents.-Kutil, Z., Skultetyova, L., Rauh, D., Meleshin, M., Snajdr, I., Novakova, Z., Mikesova, J., Pavlicek, J., Hadzima, M., Baranova, P., Havlinova, B., Majer, P., Schutkowski, M., Barinka, C. The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries.
- MeSH
- HEK293 buňky MeSH
- histondeacetylasa 6 chemie metabolismus MeSH
- katalytická doména * MeSH
- lidé MeSH
- lysin chemie metabolismus MeSH
- peptidové fragmenty chemie metabolismus MeSH
- statická elektřina MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In order to study the effects of peptide exposure to oxidative attack, we chose a model reaction in which the hydroxyl radical discretely abstracts a hydrogen atom from the α-carbon of each residue of a highly amyloidogenic region in the human calcitonin hormone, hCT15-19. Based on a combined Molecular Mechanics / Quantum Mechanics approach, the extended and folded L- and D-configuration and radical intermediate hCT15-19 peptides were optimized to obtain their compactness, secondary structure and relative thermodynamic data. The results suggest that the epimerization of residues is generally an exergonic process that can explain the cumulative nature of molecular aging. Moreover, the configurational inversion induced conformational changes can cause protein dysfunction. The epimerization of the central residue to the D-configuration induced a hairpin structure in hCT15-19, concomitant with a possible oligomerization of human calcitonin into Aβ(1-42)-like amyloid fibrils present in patients suffering from Alzheimer's disease.
- MeSH
- amyloidní beta-protein chemie MeSH
- amyloidogenní proteiny chemie MeSH
- chemické modely MeSH
- kalcitonin chemie MeSH
- lidé MeSH
- oxidace-redukce MeSH
- peptidové fragmenty chemie MeSH
- sekundární struktura proteinů MeSH
- simulace molekulární dynamiky MeSH
- stereoizomerie MeSH
- teorie funkcionálu hustoty MeSH
- termodynamika MeSH
- vodíková vazba MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The transient receptor potential channel of melastatin 4 (TRPM4) belongs to a group of large ion receptors that are involved in countless cell signalling cascades. This unique member is ubiquitously expressed in many human tissues, especially in cardiomyocytes, where it plays an important role in cardiovascular processes. Transient receptor potential channels (TRPs) are usually constituted by intracellular N- and C- termini, which serve as mediators affecting allosteric modulation of channels, resulting in the regulation of the channel function. The TRPs tails contain a number of conserved epitopes that specifically bind the intracellular modulators. Here, we identify new binding sites for the calmodulin (CaM) and S100 calcium-binding protein A1 (S100A1), located in the very distal part of the TRPM4 N terminus. We have used chemically synthesized peptides of the TRPM4, mimicking the binding epitopes, along with fluorescence methods to determine and specify CaM- and S100A1-binding sites. We have found that the ligands binding epitopes at the TRPM4 N terminus overlap, but the interacting mechanism of both complexes is probably different. The molecular models supported by data from the fluorescence method confirmed that the complexes formations are mediated by the positively charged (R139, R140, R144) and hydrophobic (L134, L138, V143) residues present at the TRPM4 N terminus-binding epitopes. The data suggest that the molecular complexes of TRPM4/CaM and TRPM4/S100A1 would lead to the modulation of the channel functions.
- MeSH
- databáze proteinů MeSH
- epitopy MeSH
- expertní systémy MeSH
- fluorescenční polarizace MeSH
- interakční proteinové domény a motivy MeSH
- kalmodulin chemie genetika metabolismus MeSH
- kationtové kanály TRPM chemie genetika metabolismus MeSH
- kinetika MeSH
- konformace proteinů MeSH
- konzervovaná sekvence MeSH
- lidé MeSH
- ligandy MeSH
- molekulární modely * MeSH
- mutace MeSH
- peptidové fragmenty chemická syntéza chemie genetika metabolismus MeSH
- proteiny S100 chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- simulace molekulového dockingu MeSH
- substituce aminokyselin MeSH
- vazebná místa MeSH
- výpočetní biologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
BACKGROUND: Neurotensin receptors are overexpressed in several cancer types including pancreatic ductal adenocarcinoma. Three NTR subtypes have been cloned: NTR-1, NTR-2 and NTR-3. The most expressed NTR-1 is not present in normal pancreatic tissue and has a low expression in chronic pancreatitis. OBJECTIVE: Objective of this study was to test in vitro affinity of the new 68Ga labelled neurotensin analogue DOTA-NT-20.3 (fragment 6-13, Ac-Lys(DOTA)-Pro-Arg(N-CH3)-Arg-Pro-Tyr-Tle-Leu) on the human pancreatic ductal adenocarcinoma cell line AsPC-1. METHOD: For the preparation of 68Ga-DOTA-NT-20.3, 68GaCl3 solution (eluted from 68Ge/68Ga generator) and 50 μg of precursor (Iason, Graz, Austria) water dissolved were used in an automatic synthesis module. The labeled compound was added to cell culture flask and incubated at 37°C. At various time points after tracer addition up to 80min, cells were recovered, rinsed and counted for radioactivity. Results were expressed as percent binding normalized to 200000 cells and affinity parameters were calculated. RESULTS: Labeling yield was ≥98 %. The molar ratio between labelled and total peptide was about 1/400. AsPC-1 cell line showed rapid uptake of the tracer including surface and internalized binding, tending to a plateau phase 80 min after tracer addition (11%/200.000 cells). The Kd (7.335 pmol) and Bmax (90.52 kBq) value indicated high tracer affinity for AsPC-1cell line especially if compared with the literature data regarding other malignancies (e.g. colonic cancer cell line). Binding sites were 1.09x106 sites per cell. CONCLUSION: New tracer 68Ga-DOTA-NT-20.3 can be a suitable candidate for the clinical use in patients with pancreatic ductal adenocarcinoma.
- MeSH
- adenokarcinom metabolismus patologie MeSH
- heterocyklické sloučeniny monocyklické chemie metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory slinivky břišní metabolismus patologie MeSH
- neurotensin agonisté chemie metabolismus MeSH
- peptidové fragmenty chemie metabolismus MeSH
- radioizotopy galia chemie metabolismus MeSH
- receptory neurotensinu metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Multi-target drug discovery is one of the most followed approaches in the active central nervous system (CNS) therapeutic area, especially in the search for new drugs against Alzheimer's disease (AD). This is because innovative multi-target-directed ligands (MTDLs) could more adequately address the complexity of this pathological condition. In a continuation of our efforts aimed at a new series of anti-AD MTDLs, we combined the structural features of the cholinesterase inhibitor drug tacrine with that of resveratrol, which is known for its purported antioxidant and anti-neuroinflammatory activities. The most interesting hybrid compounds (5, 8, 9 and 12) inhibited human acetylcholinesterase at micromolar concentrations and effectively modulated Aβ self-aggregation in vitro. In addition, 12 showed intriguing anti-inflammatory and immuno-modulatory properties in neuronal and glial AD cell models. Importantly, the MTDL profile is accompanied by high-predicted blood-brain barrier permeability, and low cytotoxicity on primary neurons.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- Alzheimerova nemoc farmakoterapie metabolismus MeSH
- amyloidní beta-protein chemie MeSH
- antioxidancia chemie metabolismus farmakologie terapeutické užití MeSH
- butyrylcholinesterasa metabolismus MeSH
- cholinesterasové inhibitory chemie metabolismus farmakologie terapeutické užití MeSH
- cílená molekulární terapie * MeSH
- hematoencefalická bariéra metabolismus MeSH
- játra účinky léků MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- ligandy MeSH
- neuroprotektivní látky chemie farmakologie terapeutické užití MeSH
- peptidové fragmenty chemie MeSH
- proteinové agregáty účinky léků MeSH
- racionální návrh léčiv * MeSH
- stilbeny chemie MeSH
- takrin chemie metabolismus farmakologie terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
