|
|
The Role of Lipid Rafts in Translocation of the Adenylate Cyclase Toxin of B. pertussis across Cytoplasmatic Membrane
Chvojková, Věra ; Šebo, Peter (advisor) ; Gahura, Ondřej (referee)
The bacterium Bordetella pertussis is the causative agent of pertussis or whooping cough. The main virulent factor of this gram-negative bacterium is adenylate cyclase toxin, a member of the RTX protein family. After secretion, the toxin binds to CD11b/CD18 integrin receptor of myeloid phagocytic cells and consequently translocates its adenylate cyclase enzyme (AC) into the cytosol of the host cells. In the cytosol, the AC is activated by calmodulin, followed by rapid conversion of ATP into the signaling molecule cAMP, resulting in paralysis of bactericidal functions of phagocytic cells. Recently it was shown that translocation of the catalytic AC domain into the cytosol proceeds in two steps. After binding of CyaA to the receptor, the influx of calcium ions into the cell occurs. High local concentration of calcium induces the translocation of the CyaA- CD11b/CD18 complex into the lipid raft, where the translocation of adenylate cyclase enzyme into cytosol occurs. This work is aiming on the description of the new established paradigm dealing with membrane translocation of RTX toxins.
|
|
|
Regulatory roles of PAG and CSK in FcɛRI signaling of mast cells
Potůčková, Lucie ; Dráber, Petr (advisor) ; Šebo, Peter (referee) ; Holáň, Vladimír (referee)
8 1 ABSTRACT (EN) This thesis is focused mainly on understanding mechanisms of regulatory roles of C-terminal Src kinase (CSK) and phosphoprotein associated with glycosphingolipid- enriched microdomains (PAG) in the high-affinity IgE receptor (FcɛRI)-mediated signaling of murine mast cells. FcɛRI activation is initiated by aggregation of the receptor by complexes of multivalent antigen with IgE, followed by activation and enhanced activities of protein tyrosine kinases, phosphatases, adaptor proteins and number of other signal transduction molecules. The signaling events result in mast cell degranulation and release of variety of proinflammatory mediators, responsible for initiation of allergy and other inflammatory diseases. Understanding the function of key regulatory molecules controlling FcεRI-mediated mast cell activation, degranulation, and cytokines production could have therapeutic impact. CSK is a major negative regulator of Src family tyrosine kinases (SFKs) that play a critical role in various immunoreceptor signaling events. However, its function in mast cell activation has not been completely understood. Because of its cytoplasmic localization, CSK was assumed to be brought to the vicinity of the plasma membrane- bound SFKs via binding to membrane-bound adaptors and PAG was a major candidate....
|
|
| |
|
|
Preparation, expression and characterization of mouse GCPIII
Bäumlová, Adriana ; Konvalinka, Jan (advisor) ; Šebo, Peter (referee)
English abstract Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a type II transmembrane glycoprotein which has been discovered in nervous system as an enzyme responsible for the hydrolysis of neuropeptide N-acetyl-L-aspartyl-L-glutamate to N-acetyl-L-aspartate and L-glutamate and that has been hypothesized to influence glutamatergic signaling processes. Except for brain, GCPII was mainly found in prostate, kidney, and small intestine. In small intestine, GCPII cleaves terminal glutamates from polyglutamylated folates facilitating thus absorption of dietary folates. In prostate, this enzyme is known as prostate-specific membrane antigen and is used as a cancer marker. Mus musculus is an important model for studing GCPII and its homologs as a therapeutic target. While human GCPII and its paralog GCPIII are relatively well characterized, no biochemical study of their mouse orthologs is available. That is why mouse glutamate carboxypeptidase III (mGCPIII) was cloned, prepared by recombinant expression in insect cells and characterized. We show that pure mouse GCPIII possesses α-glutamate carboxypeptidase activity which is effectively inhibited by specific inhibitor GCPII, 2-PMPA. We also analyzed sensitivity and specifity of monoclonal antibodies against mouse GCPIII. Immunoblots demonstrate that...
|
|
|
Topography of signaling molecules on the plasma membrane in the course of mast cell activation
Lebduška, Pavel ; Dráber, Petr (advisor) ; Šebo, Peter (referee) ; Benada, Oldřich (referee) ; Tučková, Ludmila (referee)
presented technique of plasma membrane sheet preparation from nonadherent cells may facilitate research in this field. It must be, however, mentioned that a plastic view ofsignal transduction across the plasma membrane can be achieved only by combination of various mutually complementary approaches. Conclusions Three techniques of lsoladon of plasmr m€mbrane sh$ts from nonadherent BMMC mast cells have bmn developed. one of them, based on edsorption ofl€ukocýes to glass suďace, turned out to be very promlsing md provided many scientifrc datr(article E). Actlvation of RBL m9st c€l|s by FGRI raeptor dimerintion led to increme of Grb2 adaptor content in the Plasma membrane. Ilowever' by contřast to the case of receptor mu|t|merintion, this Grb2 did not sign|ficantly colocallz€ w|th FERI' and' by |mmuno|rbeling of membrane she€ts, distribution of FC5RI wrs not d|stinguishabl€ from the disfibution on nonact|vrted ce|ls (article A). BMMC, In contrast to RBL cells, after multimerization of FaRI did not form larger aggregat€s ofthis r€c€ptor thrn nonact|vat€d cells did. FGRI muldmer|ation led to lts int€rna|iation of comparable intensity rnd overa|l dynemics ln BMMC end RBL cel|s' but loce| redistribut|on of FaRI fundamentďly differed betwcn these two c€|| wes (article E). Established mode| oflrrg€ (8pproxim8t€|y...
|
|
| |
|
|
Interaction of the adenylate cyclase toxin with complement receptor 3 - Relation of structure and function
Morová, Jana ; Šebo, Peter (advisor) ; Konvalinka, Jan (referee) ; Bezouška, Karel (referee)
4 CoNcl-usroxs PnonucrroN oF tNrEcRtN CDI lb/CDlg F Four fragments of the subunit cDilb were produced and purified and then they were used for immunization of mice and for preparation of specific antibodíes. detected that 52 cells are not able to transport effectively this subunit to cell surface or to secrete its extracellular domain into medium and not even it has a signal peptide specific for 52 cells. Further it has been shorvn, that the level of production of CDl8 subunit was not affected by usage of constitutive or inducible plasmid. ANALYSIS oF THE |NTERÁCTIoN oF RTx ToxINs w|TH p2 INTEGRINS - THE RoLE oF THT, GLYCOSYLATION OF RECEPTORS IN BINDINC ON RTX TOXIN of cell surface glycoproteins, suggesting that cyaA binding to the cell surface- expressed cDllb/cDl8 integrin fully depends on its glycolsylation. It has been a|so demonstrated. that the deglycosylation did not affected ťormation of CD I I b/CD l8 heterodimer or its expression to cell surface. inhibited in the presence ofonly saccharide units that occur in the oligosaccharide chain of integrin molecule. This demonstrates, that cyaA directry recognizes the N-linked oligosaccharide chain ofits p2 integrin recepror. CD l I b/CD I 8-expressing cells. cytotoxic activity of others RTX toxins. l5...
|
|
|
Virulence of Bordetella pertussis from an Omics Perspective
Novák, Jakub ; Šebo, Peter (advisor) ; Černý, Jan (referee) ; Novák, Petr (referee)
The Gram-negative aerobic coccobacillus Bordetella pertussis is one of the few exclusively human pathogens and the main causative agent of the respiratory infectious disease called pertussis, or whooping cough. Despite global vaccination programs, pertussis remains an important public-health burden and still accounts for over 100,000 infant deaths and over a dozen of millions of whooping cough cases every year. Substantial effort is devoted to studies on the mechanisms of action of virulence factors of B. pertussis, but the biology of interactions of B. pertussis with its human host remains largely underexplored. Evolution, genetics and adaptation of B. pertussis to the complex environment of human nasopharynx and the mechanisms enabling B. pertussis to overcome host innate and adaptive mucosal immune defenses, remain poorly understood. In such situations, unbiased exploratory omics approaches represent valuable tools for uncovering of unknown aspects of host-pathogen interactions and open the path to detailed analysis of virulence-underlying processes by mechanistic studies. In this thesis, I am presenting the results of three omics projects on B. pertussis biology that involved high-throughput proteomics. In the inital phosphoprotemics project, we analyzed the kinase signaling pathways hijacked...
|
|
| |
|
|
Phosphatidylserine and phospholipid scramblase in mast cell signaling
Smrž, Daniel ; Dráber, Petr (advisor) ; Bezouška, Karel (referee) ; Šebo, Peter (referee)
6. CONCLUSIONS 1. We found that mast cell stimulation can induce PS externalization in the absence of secretory response. 2. We identified GPI-APs as molecules whose engagement can induce sustained and reversible non-apoptotic PS externalization. 3. GPI-AP-induced PS externalization was determined as non- apoptotic and distinct from the FceRl-induced PS externalization. 4. The effect of multiple triggering on PS externalization was additive and dependent on a tlpe of stimulus and cells engaged. 5. We identifred PLSCR1 as a molecule that becomes tyrosine phosphorylated in mast cells stimulated through GPI-APs. 6. We found that the PLSCR1 tyrosine phosphorylation is not associated with mast cell secretory response' and with GPI-AP- or FceRl-induced non-apoptotic PS externalization. 7. Using confocal microscopy and electron microscopy visualization of PLSCR1 in the course of mast cell activation we found that PLSCR1: (1) is not co-localized with extemalized PS, (2) is not co- localized with aggregated Thy-l.l or FceR[, and (3) does not form self-aggregates. 8. We děřelóped a modifred one-tube semi-nested PCR-ELISA. The modified assay showed higher sensitivity and specificity than the conventional hybridization-based and a modified semi-nested- based PCR-ELISA. Due to its versatility and robustness, the...
|
guest ::
