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.

• Klíčová slova
• 17β-hydroxysteroid dehydrogenase 10 (17β-HSD10), amyloid beta (Aβ), biomolecular interactions, cyclophilin D (cypD), ionic environment, mitochondrial matrix, surface plasmon resonance (SPR),
• 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
• Názvy látek
• 17-hydroxysteroidní dehydrogenasy MeSH
• 3 (or 17)-beta-hydroxysteroid dehydrogenase MeSH Prohlížeč
• amyloidní beta-protein MeSH
• ionty MeSH
• mitochondriální proteiny MeSH
• peptidové fragmenty MeSH
• peptidylprolylisomerasa F 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.

• Klíčová slova
• S100A1, TRPM4 channel, calmodulin, fluorescence anisotropy, ligand-binding domains,
• 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
• Názvy látek
• CALM1 protein, human MeSH Prohlížeč
• epitopy MeSH
• kalmodulin MeSH
• kationtové kanály TRPM MeSH
• ligandy MeSH
• peptidové fragmenty MeSH
• proteiny S100 MeSH
• rekombinantní proteiny MeSH
• S100A1 protein MeSH Prohlížeč
• TRPM4 protein, human MeSH Prohlížeč

Na+/Ca2+ exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na+ or Ca2+ specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.

• Klíčová slova
• calcium, calcium transport, calcium-binding protein, exchanger, hydrogen exchange mass spectrometry, membrane protein, membrane transport, sodium-calcium exchange, transporter,
• MeSH
• apoproteiny chemie   genetika   metabolismus   MeSH
• archeální proteiny chemie   genetika   metabolismus   MeSH
• buněčná membrána chemie   MeSH
• cystein chemie   MeSH
• interakční proteinové domény a motivy MeSH
• inzerční mutageneze MeSH
• kinetika MeSH
• konformace proteinů MeSH
• ligandy MeSH
• Methanocaldococcus metabolismus   MeSH
• molekulární modely * MeSH
• mutace MeSH
• peptidové fragmenty chemie   genetika   metabolismus   MeSH
• pumpa pro výměnu sodíku a vápníku chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sodík metabolismus   MeSH
• stabilita proteinů MeSH
• substituce aminokyselin MeSH
• vápník metabolismus   MeSH
• vazebná místa MeSH
• vodík-deuteriová výměna MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• apoproteiny MeSH
• archeální proteiny MeSH
• cystein MeSH
• ligandy MeSH
• peptidové fragmenty MeSH
• pumpa pro výměnu sodíku a vápníku MeSH
• rekombinantní proteiny MeSH
• sodík MeSH
• vápník MeSH

Bacterial RNA polymerase (RNAP) requires σ factors to recognize promoter sequences. Domain 1.1 of primary σ factors (σ1.1) prevents their binding to promoter DNA in the absence of RNAP, and when in complex with RNAP, it occupies the DNA-binding channel of RNAP. Currently, two 3D structures of σ1.1 are available: from Escherichia coli in complex with RNAP and from T. maritima solved free in solution. However, these two structures significantly differ, and it is unclear whether this difference is due to an altered conformation upon RNAP binding or to differences in intrinsic properties between the proteins from these two distantly related species. Here, we report the solution structure of σ1.1 from the Gram-positive bacterium Bacillus subtilis We found that B. subtilis σ1.1 is highly compact because of additional stabilization not present in σ1.1 from the other two species and that it is more similar to E. coli σ1.1. Moreover, modeling studies suggested that B. subtilis σ1.1 requires minimal conformational changes for accommodating RNAP in the DNA channel, whereas T. maritima σ1.1 must be rearranged to fit therein. Thus, the mesophilic species B. subtilis and E. coli share the same σ1.1 fold, whereas the fold of σ1.1 from the thermophile T. maritima is distinctly different. Finally, we describe an intriguing similarity between σ1.1 and δ, an RNAP-associated protein in B. subtilis, bearing implications for the so-far unknown binding site of δ on RNAP. In conclusion, our results shed light on the conformational changes of σ1.1 required for its accommodation within bacterial RNAP.

• Klíčová slova
• Bacillus, RNA polymerase, molecular modeling, nuclear magnetic resonance (NMR), protein structure, transcription initiation factor,
• MeSH
• Bacillus subtilis metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• DNA bakterií chemie   metabolismus   MeSH
• DNA řízené RNA-polymerasy chemie   genetika   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• konformace nukleové kyseliny MeSH
• konformace proteinů MeSH
• konzervovaná sekvence MeSH
• molekulární modely * MeSH
• peptidové fragmenty chemie   genetika   metabolismus   MeSH
• podjednotky proteinů MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• sigma faktor chemie   genetika   metabolismus   MeSH
• stabilita proteinů MeSH
• strukturní homologie proteinů MeSH
• Thermotoga maritima enzymologie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH
• DNA řízené RNA-polymerasy MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• peptidové fragmenty MeSH
• podjednotky proteinů MeSH
• rekombinantní proteiny MeSH
• sigma faktor MeSH

Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5) activates p38 mitogen-activated protein kinase and the c-Jun N-terminal kinase in response to proinflammatory and stress signals. In nonstress conditions, ASK1 is inhibited by association with thioredoxin (TRX) which binds to the TRX-binding domain (ASK1-TBD) at the N terminus of ASK1. TRX dissociates in response to oxidative stress allowing the ASK1 activation. However, the molecular basis for the ASK1:TRX1 complex dissociation is still not fully understood. Here, the role of cysteine residues on the interaction between TRX1 and ASK1-TBD in both reducing and oxidizing conditions was investigated. We show that from the two catalytic cysteines of TRX1 the residue C32 is responsible for the high-affinity binding of TRX1 to ASK1-TBD in reducing conditions. The disulfide bond formation between C32 and C35 within the active site of TRX1 is the main factor responsible for the TRX1 dissociation upon its oxidation as the formation of the second disulfide bond between noncatalytic cysteines C62 and C69 did not have any additional effect. ASK1-TBD contains seven conserved cysteine residues which differ in solvent accessibility with the residue C250 being the only cysteine which is both solvent exposed and essential for TRX1 binding in reducing conditions. Furthermore, our data show that the catalytic site of TRX1 interacts with ASK1-TBD region containing cysteine C200 and that the oxidative stress induces intramolecular disulfide bond formation within ASK1-TBD and affects its structure in regions directly involved and/or important for TRX1 binding.

• Klíčová slova
• TRX, ASK1, cysteine, disulfide bond, mass spectrometry,
• MeSH
• cystein chemie   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• lidé MeSH
• MAP kinasa-kinasa-kinasa 5 chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• peptidové fragmenty chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• thioredoxiny chemie   genetika   metabolismus   MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• MAP kinasa-kinasa-kinasa 5 MeSH
• MAP3K5 protein, human MeSH Prohlížeč
• peptidové fragmenty MeSH
• rekombinantní proteiny MeSH
• thioredoxiny MeSH
• TXN protein, human MeSH Prohlížeč

The asparaginyl endopeptidase legumain that is overexpressed in M2-polarized tumor-associated macrophages has been identified as a suitable target for elimination of these cells supporting tumor progression. To enhance the efficacy of DNA immunization against legumain, we performed several modifications in this protein that could improve induction of immune responses. First, we mutated the RGD motif into GGD or RGG sequences. This alteration resulted in diminished maturation of legumain and impaired cellular localization. Then, as tolerance to self-antigens can be broken by the activation of CD4 T-cell help, we tried to enhance the immunogenicity of legumain by the insertion of a foreign helper epitope, namely the p30 epitope from the tetanus toxin. Finally, the 2 modifications were combined. After gene gun DNA immunization of C57BL/6 mice with these constructs, we identified the Lgmn111-119 CD8 T-cell epitope that binds to H-2D molecules. Furthermore, we showed that mutagenesis in the RGD motif significantly enhanced the immune response against legumain. The addition of the p30 helper epitope induced the specific production of IFN-γ by T cells, but did not significantly increase legumain-specific immunity activated after mutagenesis in the RGD motif which might be caused by simultaneous activation of a Th2 response demonstrated by the production of IL-4. However, the beneficial effect of the helper epitope on legumain-specific response was proved after the depletion of regulatory T cells by antibody against CD25 that preferentially stimulated Th1 immunity. The antitumor effect of the modified legumain gene was shown in the immunization against tumors induced by MK16 cells.

• MeSH
• aminokyselinové motivy genetika   MeSH
• biolistika MeSH
• buňky NIH 3T3 MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• cysteinové endopeptidasy genetika   metabolismus   MeSH
• DNA vakcíny * MeSH
• epitopy T-lymfocytární genetika   metabolismus   MeSH
• experimentální nádory imunologie   terapie   MeSH
• HEK293 buňky MeSH
• imunoterapie metody   MeSH
• interferon gama metabolismus   MeSH
• lidé MeSH
• makrofágy imunologie   MeSH
• mutace genetika   MeSH
• mutageneze cílená MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• peptidové fragmenty genetika   metabolismus   MeSH
• protinádorové vakcíny * MeSH
• T-lymfocyty pomocné-indukující imunologie   MeSH
• tetanový toxin genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• asparaginylendopeptidase MeSH Prohlížeč
• cysteinové endopeptidasy MeSH
• DNA vakcíny * MeSH
• epitopy T-lymfocytární MeSH
• interferon gama MeSH
• nádorové biomarkery MeSH
• P30 tetanus toxin peptide MeSH Prohlížeč
• peptidové fragmenty MeSH
• protinádorové vakcíny * MeSH
• tetanový toxin MeSH

In DNA vaccination, CD4(+) T-cell help can be enhanced by fusion of a gene encoding an immunization protein with a foreign gene or its part providing T(h) epitopes. To study the effect of helper epitope localization in a protein molecule, the influence of the vicinity of the helper epitope, and the impact of chimeric protein cellular localization, we fused the helper epitope p30 from tetanus toxin (TT, aa 947-967) with the N- or C-terminus of the mutated E7 oncoprotein (E7GGG) of human papillomavirus type 16, enlarged the p30 epitope with the flanking residues containing potential protease-sensitive sites and altered the cellular localization of the fusion constructs by signal sequences. The p30 epitope enhanced the E7-specific response, but only in constructs without added signal sequences. After localization of the fusion proteins into the endoplasmic reticulum and endo/lysosomal compartment, the TT-specific T(h)2 response was increased. The synthetic Pan DR epitope (PADRE) induced a stronger E7-specific response than the p30 epitope and its stimulatory effect was not limited to nuclear/cytoplasmic localization of the E7 antigen. These results suggest that in the optimization of immune responses by adding helper epitopes to DNA vaccines delivered by the gene gun, the cellular localization of the antigen needs to be taken into account.

• MeSH
• biolistika metody   MeSH
• buňky NIH 3T3 MeSH
• cytokiny metabolismus   MeSH
• DNA vakcíny aplikace a dávkování   farmakologie   MeSH
• endoplazmatické retikulum imunologie   metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• Papillomavirus E7 - proteiny genetika   metabolismus   farmakologie   MeSH
• peptidové fragmenty genetika   farmakologie   MeSH
• plazmidy aplikace a dávkování   MeSH
• rekombinantní fúzní proteiny metabolismus   farmakologie   MeSH
• tetanový toxin genetika   farmakologie   MeSH
• vakcína proti malárii farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny MeSH
• DNA vakcíny MeSH
• oncogene protein E7, Human papillomavirus type 16 MeSH Prohlížeč
• P30 tetanus toxin peptide MeSH Prohlížeč
• PADRE 45 MeSH Prohlížeč
• Papillomavirus E7 - proteiny MeSH
• peptidové fragmenty MeSH
• rekombinantní fúzní proteiny MeSH
• tetanový toxin MeSH
• vakcína proti malárii MeSH

During the last few decades, the treatment of HIV-infected patients by highly active antiretroviral therapy, including protease inhibitors (PIs), has become standard. Here, we present results of analysis of a patient-derived, multiresistant HIV-1 CRF02_AG recombinant strain with a highly mutated protease (PR) coding sequence, where up to 19 coding mutations have accumulated in the PR. The results of biochemical analysis in vitro showed that the patient-derived PR is highly resistant to most of the currently used PIs and that it also exhibits very poor catalytic activity. Determination of the crystal structure revealed prominent changes in the flap elbow region and S1/S1' active site subsites. While viral loads in the patient were found to be high, the insertion of the patient-derived PR into a HIV-1 subtype B backbone resulted in reduction of infectivity by 3 orders of magnitude. Fitness compensation was not achieved by elevated polymerase (Pol) expression, but the introduction of patient-derived gag and pol sequences in a CRF02_AG backbone rescued viral infectivity to near wild-type (wt) levels. The mutations that accumulated in the vicinity of the processing sites spanning the p2/NC, NC/p1, and p6pol/PR proteins lead to much more efficient hydrolysis of corresponding peptides by patient-derived PR in comparison to the wt enzyme. This indicates a very efficient coevolution of enzyme and substrate maintaining high viral loads in vivo under constant drug pressure.

• MeSH
• buněčné linie MeSH
• genové produkty gag - virus lidské imunodeficience genetika   MeSH
• genové produkty pol - virus lidské imunodeficience genetika   MeSH
• geny gag MeSH
• geny pol MeSH
• HEK293 buňky MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 genetika   izolace a purifikace   fyziologie   MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy terapeutické užití   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• peptidové fragmenty genetika   MeSH
• virová léková rezistence genetika   MeSH
• virová nálož MeSH
• vysoce aktivní antiretrovirová terapie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• gag protein p1, Human immunodeficiency virus MeSH Prohlížeč
• genové produkty gag - virus lidské imunodeficience MeSH
• genové produkty pol - virus lidské imunodeficience MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• p2 gag peptide, Human immunodeficiency virus 1 MeSH Prohlížeč
• peptidové fragmenty MeSH

BACKGROUND: The Nse1, Nse3 and Nse4 proteins form a tight sub-complex of the large SMC5-6 protein complex. hNSE3/MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and the Nse4 kleisin subunit is related to the EID (E1A-like inhibitor of differentiation) family of proteins. We have recently shown that human MAGE proteins can interact with NSE4/EID proteins through their characteristic conserved hydrophobic pocket. METHODOLOGY/PRINCIPAL FINDINGS: Using mutagenesis and protein-protein interaction analyses, we have identified a new Nse3/MAGE-binding domain (NMBD) of the Nse4/EID proteins. This short domain is located next to the Nse4 N-terminal kleisin motif and is conserved in all NSE4/EID proteins. The central amino acid residues of the human NSE4b/EID3 domain were essential for its binding to hNSE3/MAGEG1 in yeast two-hybrid assays suggesting they form the core of the binding domain. PEPSCAN ELISA measurements of the MAGEC2 binding affinity to EID2 mutant peptides showed that similar core residues contribute to the EID2-MAGEC2 interaction. In addition, the N-terminal extension of the EID2 binding domain took part in the EID2-MAGEC2 interaction. Finally, docking and molecular dynamic simulations enabled us to generate a structure model for EID2-MAGEC2. Combination of our experimental data and the structure modeling showed how the core helical region of the NSE4/EID domain binds into the conserved pocket characteristic of the MAGE protein family. CONCLUSIONS/SIGNIFICANCE: We have identified a new Nse4/EID conserved domain and characterized its binding to Nse3/MAGE proteins. The conservation and binding of the interacting surfaces suggest tight co-evolution of both Nse4/EID and Nse3/MAGE protein families.

• MeSH
• chromozomální proteiny, nehistonové MeSH
• inhibitor diferenciace 2 MeSH
• interakční proteinové domény a motivy MeSH
• intracelulární signální peptidy a proteiny chemie   genetika   metabolismus   MeSH
• jaderné proteiny chemie   MeSH
• konzervovaná sekvence MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• peptidové fragmenty chemie   genetika   metabolismus   MeSH
• počítačová simulace MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• Schizosaccharomyces pombe - proteiny chemie   MeSH
• Schizosaccharomyces MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• techniky dvojhybridového systému MeSH
• transportní proteiny chemie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromozomální proteiny, nehistonové MeSH
• EID2 protein, human MeSH Prohlížeč
• EID3 protein, human MeSH Prohlížeč
• inhibitor diferenciace 2 MeSH
• intracelulární signální peptidy a proteiny MeSH
• jaderné proteiny MeSH
• Nse3 protein, S pombe MeSH Prohlížeč
• Nse4 protein, S pombe MeSH Prohlížeč
• NSMCE3 protein, human MeSH Prohlížeč
• peptidové fragmenty MeSH
• proteiny buněčného cyklu MeSH
• rekombinantní proteiny MeSH
• Schizosaccharomyces pombe - proteiny MeSH
• SMC5 protein, human MeSH Prohlížeč
• transportní proteiny MeSH

Human CMV infects between 50-85% of healthy individuals and can cause live-threatening infections in immunocompromised patients. Therefore, peptide vaccination is being developed as a promising immunotherapeutic approach for treatment of patients at risk of CMV disease. The enzymatically inactive toxoid of Bordetella adenylate cyclase (CyaA-AC(-)) was shown to be an efficient tool for delivery of peptide epitopes and stimulation of Ag-specific T-cell immune responses. We investigated here the capacity of two CyaA-AC(-) constructs to deliver epitopes derived from the CMV phosphoprotein pp65 for activation of human T cells in vitro. Expansion of γ-IFN-secreting CMV-specific CD8(+) T cells, as well as increase of total IFN-γ and TNF-α production by PBMCs from CMV-seropositive donors were observed after in vitro stimulation with CyaA-AC(-) constructs carrying CMV epitopes, whereas limited activation of immune response occurred with free peptides. The activation of immune response was confirmed by expansion of CMV-specific T-cell clones and anti-CMV cytotoxic effect of stimulated PBMCs. These data open the way to clinical evaluation of CyaA-AC(-) constructs as tools for detection and expansion of CMV-specific T-cell immune responses for diagnostic and immunotherapeutic applications against CMV-associated diseases.

• MeSH
• adenylátcyklasy genetika   imunologie   MeSH
• aktivace lymfocytů MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• Cytomegalovirus imunologie   MeSH
• epitopy T-lymfocytární imunologie   MeSH
• fosfoproteiny imunologie   MeSH
• lidé MeSH
• peptidové fragmenty genetika   imunologie   MeSH
• proteiny virové matrix imunologie   MeSH
• sekvence aminokyselin MeSH
• vakcíny proti cytomegalovirové infekci genetika   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy MeSH
• cytomegalovirus matrix protein 65kDa MeSH Prohlížeč
• epitopy T-lymfocytární MeSH
• fosfoproteiny MeSH
• peptidové fragmenty MeSH
• proteiny virové matrix MeSH
• vakcíny proti cytomegalovirové infekci MeSH