|
|
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...
|
|
|
The role of ORMDL proteins in mast cell signaling
Paulenda, Tomáš ; Dráber, Petr (advisor) ; Heneberg, Petr (referee) ; Malý, Petr (referee)
1. Abstract (EN) This thesis is collection of work focused mainly on the understanding of mast cell activation and its regulation by Orm1-like (ORMDL) proteins. ORMDL family is a group of endoplasmic reticulum (ER) membrane resident proteins that are highly conserved amongst mammalian species. ORMDL proteins can be found in diverse range of organisms from plants through fungi to animals. ORMDL proteins were first discovered in yeasts and the interest in these proteins skyrocketed after the discovery that ORMDL3 is associated with childhood onset asthma in genome wide association studies. Following research connected ORMDL3 also with allergic inflammation and inflammatory bowel disease. Since mast cells are mainly known for their role in allergy and allergen induced inflammation, we decided to investigate the role of ORMDL proteins in regulation of mast cell activation and signaling. In our first study we focused on the role of ORMDL3 in mast cell activation via the high affinity IgE receptor 1 (FcεRI). We prepared bone marrow-derived mast cells with decreased (ORMDL3-KD) or increased (ORMDL3-OE) ORMDL3 expression. We showed that ORMDL3 is a negative regulator of mast cell activation events like degranulation, cytokine release and migration, without any effect on calcium mobilization. ORMDL3 was previously...
|
|
|
Involvement of Asthma-associated Protein ORMLDL3 in Mast Cell Signalling
Eitler, Jiří ; Dráber, Petr (advisor) ; Konvalinka, Jan (referee)
4 Abstract Mast cells are involved in variety of immunological processes, but they are mostly known for their role in allergy and asthma. As asthma and allergy are serious diseases with spreading tendency during last decades, mast cells are subject of intensive research. It is expected that studies of mast cell signalling pathways will contribute to our understanding of the nature of these diseases and help to design efficient treatment strategies. In an attempt to identify genes responsible for asthma disease, genome-wide screening methods have been currently applied. Using these methods, mutations in ORMDL3 (Orosomucoid1-like) protein were found out as a high risk asthma factor. ORMDL3 is a member of evolutionary conserved ORMDL family, comprising in mammals also of ORMDL1 and ORMDL2. Physiological function of these proteins is poorly understood and it has not been studied in mast cells. We decided to study the role of ORMDL proteins in mast cells. Lentiviral delivery system was optimised for generation of stable knock-downs (KD) of all three members of the ORMDL family in primary mast cells. The ORMDL gene expression was measured by improved qPCR (quantitative PCR) reaction buffers. We found that all ORMDL genes are expressed in mast cells in order ORMDL3 > ORMDL2 > ORMDL1. Next, we investigated the...
|
|
|
The role of ORMDL proteins in mast cell signaling
Paulenda, Tomáš ; Dráber, Petr (advisor) ; Heneberg, Petr (referee) ; Malý, Petr (referee)
1. Abstract (EN) This thesis is collection of work focused mainly on the understanding of mast cell activation and its regulation by Orm1-like (ORMDL) proteins. ORMDL family is a group of endoplasmic reticulum (ER) membrane resident proteins that are highly conserved amongst mammalian species. ORMDL proteins can be found in diverse range of organisms from plants through fungi to animals. ORMDL proteins were first discovered in yeasts and the interest in these proteins skyrocketed after the discovery that ORMDL3 is associated with childhood onset asthma in genome wide association studies. Following research connected ORMDL3 also with allergic inflammation and inflammatory bowel disease. Since mast cells are mainly known for their role in allergy and allergen induced inflammation, we decided to investigate the role of ORMDL proteins in regulation of mast cell activation and signaling. In our first study we focused on the role of ORMDL3 in mast cell activation via the high affinity IgE receptor 1 (FcεRI). We prepared bone marrow-derived mast cells with decreased (ORMDL3-KD) or increased (ORMDL3-OE) ORMDL3 expression. We showed that ORMDL3 is a negative regulator of mast cell activation events like degranulation, cytokine release and migration, without any effect on calcium mobilization. ORMDL3 was previously...
|
|
|
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...
|
|
| |
|
|
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...
|
|
| |
|
|
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....
|
|
|
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...
|
guest ::
RSS feed.