UNIVERSITÄTSKLINIKUM HAMBURG-EPPENDORF Zentrum für Diagnostik / Institut für Neuropathologie Direktor Prof. Dr. med. Markus Glatzel An investigation of interaction between Neuroserpin and its putative targets PC1/3, PC2 and Furin with a YFP-protein complementation assay Dissertation zur Erlangung des Grades eines Doktors der Medizin an der Medizinischen Fakultät der Universität Hamburg. vorgelegt von: Felix Johannes Alfred Wiesmüller aus Regensburg Hamburg 2015 (wird von der Medizinischen Fakultät ausgefüllt) Angenommen von der Medizinischen Fakultät der Universität Hamburg am: 07.12.2015 Veröffentlicht mit Genehmigung der Medizinischen Fakultät der Universität Hamburg. Prüfungsausschuss, der/die Vorsitzende: Prof. Dr. Markus Glatzel Prüfungsausschuss, zweite/r Gutachter/in: Prof. Dr. Hans-Jürgen Kreienkamp Prüfungsausschuss, dritte/r Gutachter/in: 2 Index 1. Introduction ....................................................................................................................... 5 1.1. Neuroserpin .................................................................................................................. 5 1.1.1. Discovery .................................................................................................... 5 1.1.2. Serpin family and serpin kinetics ................................................................ 6 1.1.3. Neuroserpin gene and regulation of expression .......................................... 9 1.1.4. 3D crystal structure ................................................................................... 11 1.1.5. Tissue distribution ..................................................................................... 12 1.1.6. Cellular localization and trafficking ......................................................... 13 1.1.7. Known targets ........................................................................................... 14 1.1.8. Known functions ....................................................................................... 16 1.1.8.1. Role in neurogenesis ...................................................................... 16 1.1.8.2. Role in regeneration ...................................................................... 16 1.1.8.3. Role in neuroendocrine system ..................................................... 17 1.1.8.4. Role in synaptic plasticity ............................................................. 17 1.1.8.5. Role in the regulation of the emotional state ................................. 18 1.1.8.6. Neuroprotective role ...................................................................... 18 1.1.9. Involvement in pathogenesis ..................................................................... 19 1.1.9.1. Alzheimer’s disease ....................................................................... 19 1.1.9.2. Cancer ............................................................................................ 19 1.1.9.3. Schizophrenia ................................................................................ 20 1.1.9.4. FENIB ............................................................................................ 20 1.2. Tissue Plasminogen Activator .................................................................................... 22 1.2.1. General aspects ......................................................................................... 22 1.2.2. Pathological aspects .................................................................................. 24 1.2.2.1. Addiction ....................................................................................... 24 1.2.2.2. Alzheimer’s disease ....................................................................... 24 1.2.2.3. Stroke ............................................................................................. 25 1.3. Proprotein convertases ............................................................................................... 26 1.3.1. PC1/3 ......................................................................................................... 26 1.3.2. PC2 ............................................................................................................ 27 1.3.3. Furin .......................................................................................................... 29 1.4. YFP-PCA ................................................................................................................... 31 2. Material and Methods ..................................................................................................... 34 2.1. YFP-tag constructs ..................................................................................................... 34 2.2. Subcloning of POMC cDNA ..................................................................................... 34 2.3. Cell culture and DNA transfection ............................................................................. 35 2.4. FACS sorting .............................................................................................................. 36 2.5. Fluorescence Microscopy ........................................................................................... 36 2.6. SDS-PAGE ................................................................................................................. 37 2.7. Animals ...................................................................................................................... 38 2.8. Pituitary protein extracts ............................................................................................ 38 2.9. Brain extracts ............................................................................................................. 39 2.10. Statistics ................................................................................................................... 39 2.11. Antibodies ................................................................................................................ 40 3 3. Results .............................................................................................................................. 41 3.1. Testing successful transfection and localization of Neuroserpin and targets by Immunofluorescence ............................................................................................. 41 3.2. Testing complex formation by YFP-PCA in HEK and N2A cells ............................. 44 3.3. Testing complex formation by YFP-PCA in cells from adrenal medulla .................. 46 3.4. Investigation of complex formation by Western blot analysis ................................... 48 3.5. Enrichment of neuroserpin-PC2-cotransfected cells for WB analysis ....................... 52 3.6. Analysis of inhibition of PC2 proteolytic activity by neuroserpin ............................ 54 3.7. Expression of PC2 inversely correlates with expression of neuroserpin in murine pituitary ...................................................................................................... 56 3.8. Expression and activity of PC2 in wild-type and neuroserpin knock-out murine brain extract ................................................................................................... 57 4. Discussion .......................................................................................................................... 60 4.1. YFP-protein fragment complementation assay .......................................................... 61 4.2. No evidence for complex formation between tPA and neuroserpin in YFP-PCA ................................................................................................................... 62 4.3. PC1/3 does not undergo complex formation with neuroserpin .................................. 63 4.4. Furin does not interact with neuroserpin, but in the presence of Furin neuroserpin undergoes complex formation with an unidentified protein ........................................................................................................................ 63 4.5. PC2 undergoes complex formation with neuroserpin ................................................ 64 4.6. Does neuroserpin inhibit PC2 in its proteolytic activity? .......................................... 65 4.7. Implications ................................................................................................................ 66 5. Synopsis ............................................................................................................................ 67 6. Appendix .......................................................................................................................... 68 6.1. Abbreviations ............................................................................................................. 68 6.2. List of figures ............................................................................................................. 73 6.3. List of tables ............................................................................................................... 75 7. References ........................................................................................................................ 76 8. Acknowledgments ............................................................................................................ 86 9. Curriculum vitae ............................................................................................................. 87 10. Eidesstattliche Erklärung ..............................................................................................