Natural killer (NK) cells play a significant role in the detection and destruction of virally infected and tumor cells. The NKR-P1 receptors regulate NK cell function by an alternative missing-self recognition system. Although the NKR-P1 receptors were among the first surface NK receptors identified on rodent NK cells more than 20 years ago, there is still very little known about their biological function and their physiological ligands. Furthermore, no three-dimensional structure of any of the NKR-P1 family receptors has been published so far. To understand the functional architecture of mouse NKR-P1 receptors, we developed a simple and efficient protocol providing large amounts of pure soluble NKR-P1 proteins. The crystal structure of mouse NKR-P1A, determined at 1.7 A resolution, is the first structure of a representative of the NKR-P1 family. Crystal structure is formed by a compact C-type lectin-like domain and an extended loop that participates in domain swapping. A potential role of the swapped loop has been suggested in natural ligand binding by in silico studies. However, chemical cross-linking and H/D exchange in combination with high resolution mass spectrometry revealed this loop in close proximity to the compact core in solution. The discrepancy between the crystal and solution structure...

It has recently been discovered that the effect of morphine can significantly reduce the tissue damage that occurs during myocardial ischemia. The molecular mechanisms by which morphine acts on the heart are still little understood. The aim of this thesis was to monitor the effect of chronic 27-day and 10-day administration of low (1 mg/kg/day) and high (10 mg/kg/ day) doses of morphine on the expression of selected G-protein-coupled receptors (GPCR) and on the expression and activity of adenylyl cyclase (AC). Chronic (27 days) morphine treatment reduced the expression of к-opioids receptors, but 10-day morphine exposure did not influence the expression of these receptors. Assessment of β1- and β2-AR by immunoblot technique did not show any significant change in the expression, but the more accurate determination of β-AR expression using the saturation binding studies revealed that 27-day treatment with high doses of morphine appreciable increased the total number of these receptors. Administration of high doses of morphine led to marked up-regulation of adenylyl cyclase (AC) isoforms V/VI, and the amount of AC decreased proportionally with the time of discontinuation of morphine administration. Low doses of morphine up- regulated AC only during 27-day administration. Chronic morphine exposure did...

2. Abstract Adaptation to chronic hypoxia is in addition to ischemic preconditioning one of the two known cardioprotective mechanisms. The precise molecular basis of these processes is still not fully explained. There are some studies that suggest the possible involvement of the adenosinergic signaling system in this adaptation. In this work, we focused on the characterization of the adenosinergic system in the myocardium of rats adapted to two regimens of chronic hypoxia - a protective continuous normobaric hypoxia (CNH) and non-protective intermittent hypoxia (INH/R, 23 h hypoxia and 1 h normoxia). Initially, we compared the total amount of adenosine receptors in samples from different groups of adapted animals. We discovered changes mainly at A2B receptor, which increased at CNH and declined in INH/R. This result suggests the possible involvement of A2B receptors in cardioprotection afforded by adaptation to chronic hypoxia. Furthermore, we investigated the distribution of various types of adenosine receptors and transporters in the plasma membrane of cardiac cells. We observed that A2A and A3 localize in membrane microdomains together with membrane enzyme CD73 that produces adenosine in the extracellular space by degrading AMP. A1 and A2B receptors similarly as nucleoside transporters ENT1, ENT2 and...

Adenosine plays a critical role in the heart signalling while affecting heart rate, contractility or coronary flow. Nowadays, four adenosine receptor subtypes are distinguished which are present in most of tissues and cells: A1, A2A, A2B and A3. All these receptors belong to the family of G protein-coupled receptors. Upon activation, their main target is an enzyme adenylyl cyclase which produces an important second messenger cAMP. The main goal of this thesis was characterization of adenosine receptors in the rat myocardium, assessment of their distribution, binding properties and signalling. We examined a possible disparity in receptors distribution between the left and right ventricles using SDS-PAGE electrophoresis and Western blotting. The same methods have been used in studies of adenosine receptor distribution in lipid rafts. Samples of lipid rafts and soluble fraction were prepared using a nonionic detergent Triton X-100. We did not find any evidence of different distribution between the left and right ventricles and our results did not confirm compartmentation of the receptors either. For determination of binding properties of the receptors we used radioligand binding assays with the A1 selective radioligand [H3 ]DPCPX. We did not observe any significant difference between the receptor...

TRAIL (TNF-related apoptosis inducing ligand) became known for its ability to selectively eliminate cancer cells. This ligand is a member of the TNF (tumor necrosis factor) ligands family and triggers extrinsic apoptotic pathway by binding of its death receptor 4 or 5 (DR4/5), and subsequent formation of death-inducing signalling complex (DISC). This signalling complex is required for successful transmission of apoptotic signal and activation of proximal caspases. However, regulation of the initial steps leading to activation of caspases is still not fully understood. Endocytosis of a TRAIL- DR4/5-DISC complex can be one of modulators of the initiation of extrinsic apoptotic pathway. Recent studies show controversial data documenting that endocytosis of TRAIL receptosomes can in cell type specific manner either positively or negatively influence TRAIL-induced apoptotic signalling. In this study, we focus on the analysis of a role of endocytosis and acidification of endosomal compartments during TRAIL-induced apoptosis in human colorectal cancer cell lines. Our results support the view that both clathrin-dependent endocytosis of TRAIL receptosome and endosomal acidification positively affect activation of caspases during the early stages of TRAIL-induced apoptosis. Inhibition of endocytosis or endosomal...

No description available.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of TNF family expressed mainly by hematopoietic cells. TRAIL brought significant attention mainly for its ability to trigger apoptosis in a number of cancer cells. In addition to apoptosis, TRAIL can induce several other signaling pathways such as activation of MAP kinases or canonical NF-B signaling. Human TRAIL can bind to five receptors but only two of them (death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5) can trigger TRAIL-mediated apoptotic and non-apoptotic signaling in target cells. Both receptors are ubiquitously expressed on normal and cancer cells, but the relative contribution of DR4 and DR5 to TRAIL-induced signaling is not well known. Using DR4/DR5-specific variants of TRAIL, we examined how individual receptor contributes to the induction of apoptosis and NF-B, JNK, p38, ERK1/2 and TAK1 signaling pathways in selected colorectal cells. We found that in DLD-1 cells, apoptosis and activation of JNKs are mainly mediated by DR4-selective ligand. In TRAIL-resistant HT-29 cells, we show that though DISC formation and activation of caspase-8 proceeds mainly via DR4-specific signaling, activation of NF-B pathway is mainly triggered by DR5 selective ligand. In other cells and analyzed signaling pathways both receptor-specific ligands triggered very...

Charles University in Prague Faculty of Science Department of Biochemistry Studies of NK cell receptors and other proteins using recombinant expressions and mass spectrometry Summary of Ph. D. Thesis Daniel Kavan Supervisor: Prof. RNDr. Karel Bezouška, DSc. Prague 2010 Daniel Kavan Introduction Introduction NK cells and CD69 as one of their surface receptors Natural killer cells (NK cells) are the subpopulation of large granular lymfocytes, which lacks the surface receptors typical for B cells or T cells. They are characterized by the presence of NKp46 and NKp30 [Moretta L. et. al. 2002], however. They were named natural killers according to their function in the organism, as they do not need any activation and nevertheless they are able to eliminate abnormal (i. e. infected or transformed) cells from the tissue [Kiessling R. et. al. 1975]. This function is dependent on scanning the major histocompatibility complex (MHC) class I molecules of ambient cells. The resulting action (killing or not killing the target cell) is dependent on the balance of activating and inhibiting signals mediated by the NK cell surface receptors and forwarded to the specific signaling pathway [Raulet D. H. et. al. 2001]. Specificity of NK cells is not based only on one type of antigen receptor as it is in case of T and B cells,...

Muscarinic acetylcholine receptors (mAChR) belong to the family of G-protein coupled receptors. There are five subtypes of mAChR denoted M1 to M5 that are widely and differentially distributed in both the central nervous system and periphery and play an important role in many specific physiological functions. Impairment of muscarinic neurotransmission occurs in serious disorders such as Alzheimer's disease, schizophrenia or Parkinson's disease that are accompanied by cognitive decline mainly due to the disruption of M1 receptor signaling in the brain. Unfortunately, the high degree homology of the orthosteric binding site among muscarinic receptor subtypes makes it very difficult to obtain subtype- selective agonists. One of the few known selective agonists is xanomeline that preferentially activates the M1 and M4 subtypes. Xanomeline exerts unique interactions with muscarinic receptors comprising reversible binding to the orthosteric domain, and wash-resistant allosteric interaction with a secondary binding site. The basis of xanomeline functional selectivity remains largely unknown. In an attempt to probe into such mechanisms we investigated the immediate and long-term effects of xanomeline on activation of muscarinic receptors, using intact Chinese hamster ovary (CHO) cells expressing individual...

The main goal of this thesis was to optimize the current therapeutic approach using TLR agonists and anchored agonists of phagocytic receptor to treating cancer. The study is focused on searching a suitable agonist of TLR as the replacement of LPS.