Merz, Stephanie Katharina (2022)
Development and Characterization of a FKBP51FK1 Affinity Matrix for Protein-Protein Interaction Studies.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00022958
Dissertation, Erstveröffentlichung, Verlagsversion
Kurzbeschreibung (Abstract)
The FK506 binding protein 51 (FKBP51) plays a role in psychiatric diseases such as major depression and anxiety. Several studies indicated that FKBP51 is also related to cancer, chronic pain and metabolic disorders such as obesity and diabetes. However, the underlying cellular and molecular biological mechanisms are poorly understood. FKBP51 belongs to the FKBP protein family that includes several FK506 binding proteins sharing structural identities. In the last years, FKBP51 has emerged as a drug target, even though the structural similarity of FKBPs makes the development of selective FKBP ligands still challenging. Two FKBP lead compounds, SAFit1 and SAFit2, showing selectivity for FKBP51 in comparison with the biological counterpart FKBP52, were developed by the Hausch research group some years ago. Both compounds interact with the binding pocket of the FK1 domain. Unfortunately, the biological function of FKBP51 and the cellular effects of FKBP ligands are hardly understood until now. The aim of the PhD project was to gain insights into the FKBP51 protein interaction network. The identification of FKBP51 binding partners is a good starting point for elucidating cellular functions, the underlying signal transduction and signaling pathways of FKBP51. The project was focused on the enrichment of potential FKBP51 binding proteins interacting with the binding pocket of the FK1 domain. For this purpose, a FKBP51FK1 affinity matrix was developed and the affinity matrix preparation protocol was optimized. A fluorescence-based binding assay was developed to characterize the FKBP51FK1 affinity matrix. The binding pocket integrity was shown by the binding and competitive elution of a fluorescein-labeled FKBP ligand. A pull-down assay applying FKBP51FK1 as bait was developed to enrich potential FKBP51 binding proteins from cell lysates. The protein enrichment was assessed by a bottom-up proteomic approach in cooperation with SINTEF Industry in Trondheim. Another part of the PhD thesis dealt with the characterization of new FKBP ligands by competitive fluorescence polarization assays. Here, high affinity ligands showing dissociation constants in the picomolar range were discovered. In addition to that, a macrocyclic compound showing selectivity for FKBP51 in comparison with FKBP12 was identified.
| Typ des Eintrags: | Dissertation | ||||
|---|---|---|---|---|---|
| Erschienen: | 2022 | ||||
| Autor(en): | Merz, Stephanie Katharina | ||||
| Art des Eintrags: | Erstveröffentlichung | ||||
| Titel: | Development and Characterization of a FKBP51FK1 Affinity Matrix for Protein-Protein Interaction Studies | ||||
| Sprache: | Englisch | ||||
| Referenten: | Hausch, Prof. Dr. Felix ; Löwer, Prof. Dr. Alexander | ||||
| Publikationsjahr: | 2022 | ||||
| Ort: | Darmstadt | ||||
| Kollation: | 241 Seiten | ||||
| Datum der mündlichen Prüfung: | 11 April 2022 | ||||
| DOI: | 10.26083/tuprints-00022958 | ||||
| URL / URN: | https://tuprints.ulb.tu-darmstadt.de/22958 | ||||
| Kurzbeschreibung (Abstract): | The FK506 binding protein 51 (FKBP51) plays a role in psychiatric diseases such as major depression and anxiety. Several studies indicated that FKBP51 is also related to cancer, chronic pain and metabolic disorders such as obesity and diabetes. However, the underlying cellular and molecular biological mechanisms are poorly understood. FKBP51 belongs to the FKBP protein family that includes several FK506 binding proteins sharing structural identities. In the last years, FKBP51 has emerged as a drug target, even though the structural similarity of FKBPs makes the development of selective FKBP ligands still challenging. Two FKBP lead compounds, SAFit1 and SAFit2, showing selectivity for FKBP51 in comparison with the biological counterpart FKBP52, were developed by the Hausch research group some years ago. Both compounds interact with the binding pocket of the FK1 domain. Unfortunately, the biological function of FKBP51 and the cellular effects of FKBP ligands are hardly understood until now. The aim of the PhD project was to gain insights into the FKBP51 protein interaction network. The identification of FKBP51 binding partners is a good starting point for elucidating cellular functions, the underlying signal transduction and signaling pathways of FKBP51. The project was focused on the enrichment of potential FKBP51 binding proteins interacting with the binding pocket of the FK1 domain. For this purpose, a FKBP51FK1 affinity matrix was developed and the affinity matrix preparation protocol was optimized. A fluorescence-based binding assay was developed to characterize the FKBP51FK1 affinity matrix. The binding pocket integrity was shown by the binding and competitive elution of a fluorescein-labeled FKBP ligand. A pull-down assay applying FKBP51FK1 as bait was developed to enrich potential FKBP51 binding proteins from cell lysates. The protein enrichment was assessed by a bottom-up proteomic approach in cooperation with SINTEF Industry in Trondheim. Another part of the PhD thesis dealt with the characterization of new FKBP ligands by competitive fluorescence polarization assays. Here, high affinity ligands showing dissociation constants in the picomolar range were discovered. In addition to that, a macrocyclic compound showing selectivity for FKBP51 in comparison with FKBP12 was identified. |
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| Status: | Verlagsversion | ||||
| URN: | urn:nbn:de:tuda-tuprints-229587 | ||||
| Sachgruppe der Dewey Dezimalklassifikatin (DDC): | 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften 500 Naturwissenschaften und Mathematik > 540 Chemie |
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| Fachbereich(e)/-gebiet(e): | 07 Fachbereich Chemie 07 Fachbereich Chemie > Clemens-Schöpf-Institut > Fachgebiet Biochemie |
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| Hinterlegungsdatum: | 21 Dez 2022 13:19 | ||||
| Letzte Änderung: | 22 Dez 2022 07:44 | ||||
| PPN: | |||||
| Referenten: | Hausch, Prof. Dr. Felix ; Löwer, Prof. Dr. Alexander | ||||
| Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: | 11 April 2022 | ||||
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