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Studies on interactions between NKR-P1D and Clrb membrane receptors
Hanč, Pavel ; Novák, Petr (advisor) ; Brdička, Tomáš (referee)
Studies on interactions between NKR-P1D and Clrb membrane receptors Interaction between murine NKR-P1D and Clrb receptors was originally described as a novel type of "MHC class-I independent missing-self recognition" and was shown to confer protection from killing by natural killer cells.[1] However, further study brought conflicting results suggesting that NKR-P1D does not binds Clrb strongly if it does at all.[2] In order to address the issues arising from these conflicting results, we have recombinantly expressed the extracellular domains of both receptors in E. coli cells and refolded the proteins in vitro. The quality of refolding was confirmed both by determining the disulphide bonding pattern using FTMS and measuring 1 H/15 N-HSQC spectra. By means of size exclusion chromatography and analytical ultracentrifuge we were unable to provide convincing results for the interaction itself. However, using SPR technique, a weak, specific, pH-dependent interaction was observed. Interaction between the proteins in solution was immobilized using chemical cross-linking technique. Three cross-linking reagents, EDC, DSG and DSS were used. The reaction mixture was separated by means of SDS-PAGE and protein bands corresponding to dimers were digested in gel. Using FT-MS we were able to find peptides from both...
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Structural biology of complex of rat NK cell receptors NKR-P1B and Clrb
Dvorská, Anna ; Vaněk, Ondřej (advisor) ; Ingr, Marek (referee)
The Natural Killer (NK) cells have an important role in the nonspecific immunity of the or- ganism. They have the ability to identify and to kill tumor cells and cells infected by a virus without preceding sensitization by antigen. Their function is directed by the amount of sti- mulation and inhibition receptors interacting with ligands on the tumor or infected cell. This thesis focuses on the preparation and the study of the complex of rat NK cellular inhi- bition receptor NKR-P1B ("natural killer cell receptor - protein 1B") and its ligand Clrb ("C-type lectin-related ligand b"). The Clrb initiates the inhibition of NKR-P1B, meaning that if the cell express Clrb, it won't be destroyed. If the cell gets infected by the rat cytome- galovirus, it loses Clrb from its surface and its destruction is therefore no longer prevented. Cells infected with this virus defend themselves from destruction by expression of the viral gene of C-type lectin RCTL, which is a homolog of Clrb. Transient transfection of human embryonic kidney 293 cell line with simple glycosylation (HEK293S GnTI− ) was used for the recombinant preparation of the soluble form of these two receptors of the rat NK cells. The native forms of the receptors - disulfidic homo- dimers - were prepared as the fusion construct with IgG Fc (using...
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Structural studies of rat NK cell receptor NKR-P1B and its ligand Clrb
Skořepa, Ondřej ; Vaněk, Ondřej (advisor) ; Kavan, Daniel (referee)
Natural killer (NK) cells are an intensively studied part of immune system possessing unique ability to recognize and induce death of tumor and virus-infected cells without prior antigen sensitization. Their function is regulated by a fine balance of signals induced by multiple activating and inhibitory cell surface receptors and their interaction with the ligands present on the target cell. This can be illustrated on the homodimeric rat inhibitory receptor NKR-P1B and its ligand Clrb which play, besides other things, crucial role in the immunological response of NK cells to the infection with rat cytomegalovirus (RCMV), one of the most studied NK cell function model in rat model organism. During RCMV infection the target cell downregulates cell surface expression of Clrb, thus decreasing inhibitory signal transmitted through the NKR-P1B receptor to the NK cell, which would ideally lead to NK cell activation and lysis of the infected cell. However, RCMV carries a gene for "decoy" surface receptor - RCTL that mimics Clrb and thus helps to escape the immunological response of NK cells. Moreover, while this escape strategy was demonstrated in the WAG rat strain, it has been shown that the NKR-P1B homologue from SD rat strain binds only Clrb and does not recognize RCTL. Thus the SD rat strain is less...
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Structural biology of complex of rat NK cell receptors NKR-P1B and Clrb
Dvorská, Anna ; Vaněk, Ondřej (advisor) ; Ingr, Marek (referee)
The Natural Killer (NK) cells have an important role in the nonspecific immunity of the or- ganism. They have the ability to identify and to kill tumor cells and cells infected by a virus without preceding sensitization by antigen. Their function is directed by the amount of sti- mulation and inhibition receptors interacting with ligands on the tumor or infected cell. This thesis focuses on the preparation and the study of the complex of rat NK cellular inhi- bition receptor NKR-P1B ("natural killer cell receptor - protein 1B") and its ligand Clrb ("C-type lectin-related ligand b"). The Clrb initiates the inhibition of NKR-P1B, meaning that if the cell express Clrb, it won't be destroyed. If the cell gets infected by the rat cytome- galovirus, it loses Clrb from its surface and its destruction is therefore no longer prevented. Cells infected with this virus defend themselves from destruction by expression of the viral gene of C-type lectin RCTL, which is a homolog of Clrb. Transient transfection of human embryonic kidney 293 cell line with simple glycosylation (HEK293S GnTI− ) was used for the recombinant preparation of the soluble form of these two receptors of the rat NK cells. The native forms of the receptors - disulfidic homo- dimers - were prepared as the fusion construct with IgG Fc (using...
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Production and purification of recombinant receptor Clrb
Prokopová, Tereza ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
The Natural Killer (NK) cells play a vital role in the nonspecific immunity. They are capable of efficient immunity reaction without any antigen specific receptors on their surface. NK cells recognize non-self molecules and they also recognize their molecules serving as health markers, and absence of these molecules such as MHC glycoproteins on the target cell surface. The NK cells are able to recognize viral infection or tumor transformation in the organism. If natural killer cell is in contact with a cell carrying an abnormally low MHC class I glycoproteins, it will create a signal which informs the cell is infected with a virus. NK cells trigger apoptosis of the target cell without prior stimulation, proliferation and differentiation. They also promote inflammatory responses by the production of chemokines and cytokines. The response is always the interplay of activating and inhibitory signals that the cell receives from its surroundings. The latest research shows that the targeted modulation of NK cells leads to less complications in bone marrow transplantation. They can be potentially used in immunotherapy, e.g. in the treatment of autoimmune diseases. Therefore, NK cells are a highly-studied group of cells. This thesis is focused on a production of Clrb ("C- type-lectin-related protein b")....
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Studies on interactions between NKR-P1D and Clrb membrane receptors
Hanč, Pavel ; Novák, Petr (advisor) ; Brdička, Tomáš (referee)
Studies on interactions between NKR-P1D and Clrb membrane receptors Interaction between murine NKR-P1D and Clrb receptors was originally described as a novel type of "MHC class-I independent missing-self recognition" and was shown to confer protection from killing by natural killer cells.[1] However, further study brought conflicting results suggesting that NKR-P1D does not binds Clrb strongly if it does at all.[2] In order to address the issues arising from these conflicting results, we have recombinantly expressed the extracellular domains of both receptors in E. coli cells and refolded the proteins in vitro. The quality of refolding was confirmed both by determining the disulphide bonding pattern using FTMS and measuring 1 H/15 N-HSQC spectra. By means of size exclusion chromatography and analytical ultracentrifuge we were unable to provide convincing results for the interaction itself. However, using SPR technique, a weak, specific, pH-dependent interaction was observed. Interaction between the proteins in solution was immobilized using chemical cross-linking technique. Three cross-linking reagents, EDC, DSG and DSS were used. The reaction mixture was separated by means of SDS-PAGE and protein bands corresponding to dimers were digested in gel. Using FT-MS we were able to find peptides from both...
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