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Bordetella Adenylate Cyclase: Molecular mechanism of Action and Its Use for Antigen Delivery
Kamanová, Jana ; Šebo, Peter (advisor) ; Dráber, Petr (referee) ; Černý, Jan (referee)
(English) 4 SUMMARY (English) The first part of this PhD. thesis deals with molecular mechanism of action of the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA belongs to the family of RTX (Repeat-in-ToXin) proteins secreted by Gram-negative bacteria and primarily targets myeloid phagocytes, expressing the CD11b/CD18 integrin receptor (also known as αMβ2, CR3 or Mac-1). Upon binding, CyaA permeabilizes cell membranes by forming small cation-selective pores, and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Elevation of the cytosolic cAMP levels by CyaA then knocks down bactericidal functions of host innate immunity. CyaA is unique among other enzymatically active toxins in its capacity to penetrate cells directly from cell surface across the cytoplasmic membrane, without the need for endocytosis. Penetrating activity of CyaA depends on plasma membrane potential and on an intact, acylated and calcium-loaded RTX cytolysin moiety. By examining a set of 18 CyaA constructs that bear overlapping deletions within AC domain and a CD8+ OVA T-cell epitope tag, we showed that the first 371 amino-terminal residues are dispensable for the CyaA capacity to deliver a passenger OVA...
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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....
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Molecular mechanisms of regulation of FcɛRI signaling in mast cells
Bambousková, Monika ; Dráber, Petr (advisor) ; Černý, Jan (referee) ; Bilej, Martin (referee)
Mast cells are critical component of the immune system. In pathological situations, they are activated and are responsible for allergic reaction. Therefore, detail understanding of mast cell activation at molecular level is important for design of new therapies of allergic diseases. Principal transmembrane receptor of mast cells is the high-affinity Fc receptor for IgE (FcεRI). FcεRI anchors IgE on mast cell surface and upon cross-linking with multivalent antigen it becomes phosphorylated at its intracellular immunoreceptor tyrosine-based activation motifs (ITAMs). This triggers signaling cascade leading to cell degranulation and cytokine production. The antigen- mediated signaling through the FcεRI is critically dependent on interplay with intracellular protein- tyrosine kinases that phosphorylate the ITAM motifs and many other components of the signaling pathway. This study was focused on better understanding of signaling events leading to mast cell activation; emphasis was put on early activation events. First, we examined the role of protein- tyrosine phosphatases (PTP) in FcεRI phosphorylation. We found that upon antigen triggering of FcεRI, PTPs undergo inhibition by oxidation of their active site located tyrosine. Studies of plasma membrane topography of inactivated PTPs showed their...
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Signaling effects of adenylate cyclase toxin action on phagocytes
Černý, Ondřej ; Šebo, Peter (advisor) ; Černý, Jan (referee) ; Dráber, Petr (referee)
The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...
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Multiple regulatory roles of the transmembrane adaptor protein NTAL in gene transcription and mast cell physiology
Polakovičová, Iva ; Dráber, Petr (advisor) ; Vyklický, Ladislav (referee) ; Hašek, Jiří (referee)
(EN) This thesis focuses mainly on understanding of the regulatory roles of the transmembrane adaptor proteins, non-T cell activation linker (NTAL) and phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), in murine mast cell signaling. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI) activation pathways in mast cells. Studies carried out on mast cells prepared from NTAL knock-out mice have indicated that NTAL is a negative regulator of FcεRI signaling, whereas experiments performed on human mast cells and rat basophilic leukemia cells with silenced NTAL expression have suggested its positive regulatory role. To thoroughly examine the involvement of NTAL in FcεRI-mediated signaling events in mouse mast cells and to determine whether different methodologies of NTAL ablation have different physiological consequences, we utilized a broad range of assays. Using bone marrow-derived mast cells (BMMCs) as a model, we obtained cells from NTAL wild type and knock-out cells and using lentiviral delivery approach we transduced part of the wild type cells, with vector bearing NTAL shRNA or empty vector to generate NTAL knock-down cells and control cells, respectively. Comparison of all four groups of generated cells in our assays...
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Regulation of mast cell activation at the level of the high-affinity IgE receptor and STIM1
Bugajev, Viktor ; Dráber, Petr (advisor) ; Černý, Jan (referee) ; Hašek, Jiří (referee)
(EN) This thesis is focused on two important gate-keepers of mast cell signaling. The first is the complex of the high-affinity receptor for immunoglobulin E (IgE) (FcεRI) associated with Lck/Yes- related novel tyrosine kinase (Lyn), which is involved in acquired immune responses and the second is the stromal interaction molecule (STIM)1, which senses calcium levels in endoplasmic reticulum (ER) and upon depletion of ER Ca2+ stores participates in opening of the plasma membrane Ca2+ release- activated Ca2+ (CRAC) channels. Although the structure of FcεRI is known for many years and numerous molecules associated with the receptor have been described, the exact molecular mechanism of initiation and termination of the FcεRI signaling is elusive. Therefore, we evaluated the current knowledge on the molecular mechanisms of FcεRI phosphorylation with emphasis on the newly described model according to which cross-talk between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) sets the threshold for FcεRI tyrosine phosphorylation (PTK-PTP interplay model). Furthermore, we extended the knowledge about topography of active phosphatases which are prone to oxidation within the clusters of transmembrane adaptor proteins non-T cell activation linker (NTAL) and linker for activation of T...
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Regulatory functions of the transmembrane adaptor protein NTAL in activation of mast cells
Tůmová, Magda ; Dráber, Petr (advisor) ; Vyklický, Ladislav (referee) ; Hašek, Jiří (referee)
Mast cells originate from hematopoietic pluripotent stem cells present in bone marrow. They do not circulate, but are spread throughout a body and reside in all vascularized tissues. Mast cells as a part of innate immune system are also able to influence adaptive immune system and are effectors in IgE-mediated allergic and inflammatory diseases. Mast cells possess high affinity receptor for IgE (FcεRI). Aggregation of the receptor triggers signaling cascades which lead to the release of preformed and de-novo synthesized effector molecules. Early phases of mast cell activation include tyrosine phosphorylation of numerous substrates and formation of signaling assemblies (also called signalosomes). Important structural components of the signalosomes are transmembrane adaptor proteins. One of them is the transmembrane adaptor protein NTAL (Non-T cell Activation Linker), which becomes rapidly tyrosine- phosphorylated upon FcεRI triggering and serves as a positive or a negative regulator of FcεRI signaling. To be able to study fine topography of NTAL and other plasma membrane components in mouse bone marrow-derived mast cells (BMMCs), we first developed a new method of plasma membrane sheets isolation from non-adherent cells. The method is based on adsorption of the cells to ultraclean glass cover-slips,...
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Role of metabotropic glutamate receptors and their associated proteins in physiology and pathophysiology
Kumpošt, Jiří ; Blahoš, Jaroslav (advisor) ; Dráber, Petr (referee) ; Viklický, Vladimír (referee)
of the thesis Glutamate is a main excitatory neurotransmitter in the brain of mammals, which activates both ionotropic and metabotropic glutamate receptors. Ionotropic receptors are responsible for fast synaptic transmission leading to membrane depolarization and Ca2+ influx into the cell. On the other hand mGlu receptors play an important role in regulation of the transmission via heterotrimeric G-proteins and activation of various signaling pathways. Postsynaptically localized group I mGlu receptors (mGluR1, 5) together with ionotropic NMDA and AMPA receptors share common large receptor signaling complexes, or signalosome facilitating glutamate signal transductions. Individual mGluR1 splice variants are differently associated with signalosome including scaffold proteins like PSD-95 which organize postsynaptic density (PSD). Heterodimerization of different mGluR1 splice variants is a focal point of my thesis together with investigation of recently discovered protein IL1RAPL1 (interleukin-1 receptor accessory protein-like 1) and its role in organization of postsynaptic signalosome. Using biochemical, immunocytochemical and functional assays we showed heterodimers of mGluR1a/1b were expressed on the plasma membrane and that heterodimers are fully functional in the recombinant system. Next we showed...
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Functional analysis of syntaxin 16 phosphorylation using yeast as a model
Volfová, Barbora ; Entlicher, Gustav (advisor) ; Dráber, Petr (referee)
4 Abstract Mechanism of fusion of intracellular membranes in eukaryotic cells involves several protein families including soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins and Sec1/Munc-18 related proteins (SM proteins). It is known that the transport is evolutionary conserved from yeast to man. Therefore for facilitating of the research, we can use simple eukaryotes Saccharomyces cerevisiae. Mammalian SNARE protein syntaxin 16 has a yeast homologue Tlg2p which is used in this study as a model for studying affects of phosphorylation to the syntaxin 16 function. Also their binding partners, SM proteins mVps45p (mammalian) and yeast Vps45p are homologous. Phosphorylation of SNARE proteins is known as a possible way of regulation of membrane fusion. Abolishment of one of the putative phosphorylation sites in Tlg2p protein, serine 90 leads to dominant effects on the exocytic and endocytic pathways. The work presented in this study shows some phenotypes of mutants based on this phosphorylation site of protein Tlg2p. Those mutants are S90A (cannot be phosphorylated) and S90D (phosphomimetic - acid carboxyl group mimics phosphate group). It was revealed that the phosphorylation of Tlg2p protein at serine 90 or the mutation Tlg2p-S90D may play some role in protecting Tlg2p...
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