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The role of ORMDL3 in the IgE receptor signaling
Vávrová, Petra ; Bugajev, Viktor (advisor) ; Černý, Jan (referee)
ORMDL proteins are regulators of serine palmitoyltransferase (SPT) enzyme, which catalyzes the first step of sphingolipid biosynthesis. Human and murine ORMDL family consists of three members, ORMDL1, ORMDL2 and ORMDL3. Human and murine ORMDLs exhibit high similarity between their amino acid sequences. ORMDL3 expression has been linked to several diseases, such as childhood-onset asthma, Crohn's disease, rheumatoid arthritis, type 1 diabetes, and primary biliary cirrhosis. High expression of ORMDL3 has been found in macrophages, T cells, eosinophils, epithelial cells and mast cells. ORMDL3 is a negative regulator of the high affinity IgE receptor I (FcɛRI)-mediated signaling in mast cells. Mast cells have an important role in the acute phase of allergic reactions and are involved in eradication of multicellular parasites. In the first part of this thesis we determined the expression of ORMDL family in peritoneal- derived mast cells (PDMCs) from Ormdl3 knock out (KO) and wild type (WT) mice. Next we determined the roles of ORMD3 in FcɛRI-mediated signaling in these PDMCs. Furthermore, we analyzed the relationship between expression of ORMDL family and SPT complex in bone marrow-derived mast cells (BMMCs) and bone marrow-derived mast cell line (BMMCL). We transduced BMMCL with vector coding SPTLC1...
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Human myotubularin-related proteins in regulation of intracellular transport
Sixtová, Nikola ; Macůrková, Marie (advisor) ; Bugajev, Viktor (referee)
The plasmatic membrane and the endomembrane compartments are composed of a variety of lipids, including phosphatidylinositol (PtdIns) and its derivates, generally called phosphoinositides (PIs). Their presence in the memebranes is low, nevertheless important. PtdIns might be phosphorylated on the third, forth and fifth position of the inositol ring and give rise to 7 distinct forms of PIs. Although, all the membranes whitin the cell contain a range of PIs, one is always more prevalent than the others on each membrane, forming a part of their identity. Many intracellular processes, including vesicular transport, are dependent on a tigh regulation and distribution of PI levels achieved by a coordinated action of particular kinases and phosphatases. Myotubularins are proteins from a protein-tyrosin-phosphatase family with a potential to dephosphorylate the third position of the inositol ring of PtdIns(3)P, typical for early endosom, or PtdIns(3,5)P₂, typical for late endosom. Through this action, myotubularins have an ability to regulate the enodomal-lysosomal degradation pathway. Up to now, 14 human paralogs of myotubularins have been discovered and they are divided into two main subgroups of 8 catalyticly active and 6 inactive enzymes. Although the functions of the active phosphatases might have...
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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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