|
|
Analysis of the molecular pathways and interactome of FUBP3
Brychta, David ; Jirkovský, Eduard (advisor) ; Karahoda, Rona (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: David Brychta Supervisor: Assist. Prof. Eduard Jirkovský, PharmD, PhD.; Assist. Prof. Nika M. Lovšin, M.Chem., PhD Title of Diploma Thesis: Analysis of the molecular pathways and interactome of FUBP3 The far upstream element-binding protein 3 (FUBP3) has been recognized by genome-wide association studies to be associated with a higher risk of osteoporotic fracture. The knowledge about this protein and its role in bone biology is quite limited, though. Therefore, we aimed to broaden the horizons and created an overview of FUBP3 protein-protein interactors and possible pathway involvement. The interacting proteins were gathered across multiple databases. Their association with osteoporosis (OP) and bone mineral density (BMD) was assessed using an online tool - OpenTargets Platform. Twelve hits associated with either were then used for qPCR analysis to investigate the influence of FUBP3 knockout. Among FUBP3 interactors were also proteins of cytosolic membraneless organelles - stress granules (SG). Stress conditions were induced and co-localization of SG markers PABPC1 and G3BP1 was carried out using immunostaining and fluorescence microscopy. We were able to identify 75 protein interactors...
|
|
|
Analysis of the molecular pathways and interactome of FUBP3
Brychta, David ; Jirkovský, Eduard (advisor) ; Karahoda, Rona (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: David Brychta Supervisor: Assist. Prof. Eduard Jirkovský, PharmD, PhD.; Assist. Prof. Nika M. Lovšin, M.Chem., PhD Title of Diploma Thesis: Analysis of the molecular pathways and interactome of FUBP3 The far upstream element-binding protein 3 (FUBP3) has been recognized by genome-wide association studies to be associated with a higher risk of osteoporotic fracture. The knowledge about this protein and its role in bone biology is quite limited, though. Therefore, we aimed to broaden the horizons and created an overview of FUBP3 protein-protein interactors and possible pathway involvement. The interacting proteins were gathered across multiple databases. Their association with osteoporosis (OP) and bone mineral density (BMD) was assessed using an online tool - OpenTargets Platform. Twelve hits associated with either were then used for qPCR analysis to investigate the influence of FUBP3 knockout. Among FUBP3 interactors were also proteins of cytosolic membraneless organelles - stress granules (SG). Stress conditions were induced and co-localization of SG markers PABPC1 and G3BP1 was carried out using immunostaining and fluorescence microscopy. We were able to identify 75 protein interactors...
|
|
|
Characterizing DDI2 protein interaction by solution NMR
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR
|
|
|
Characterizing DDI2 protein interaction by solution NMR
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR
|
guest ::
