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An investigation of interaction between Neuroserpin and its putative targets PC1/3, PC2 and Furin with a YFP-protein complementation assay
Eine Untersuchung über die Interaktion zwischen Neuroserpin und seinen vermeintlichen Zielproteasen PC1/3, PC2 und Furin unter Verwendung eines YFP-protein complementation assay
Wiesmüller, Felix Johannes Alfred
Dokument 1.pdf (4.040 KB)
Freie Schlagwörter (Englisch):
neuroserpin , PC1/3 , PC2 , YFP , YFP-PCA , PCA , tPA , POMC , ACTH , FENIB
44.47 , 35.74 , 44.90
Glatzel, Markus (Prof. Dr.)
Tag der mündlichen Prüfung:
Kurzfassung auf Englisch:
Neuroserpin is a serine protease inhibitor of the central and peripheral nervous system. It is involved in a vast variety of physiologic as well as pathologic pathways. Physiologic functions range from regulation of neuroanatomic properties (e.g., neurogenesis, synaptogenesis and synaptic plasticity) to psychologic properties (e.g., regulation of the emotional state). Neuroserpin is neuroprotective in ischemic brain injury and epilepsy, yet promotes brain metastasis. Most importantly, mutations in the neuroserpin gene lead to a disease called FENIB via accumulation of neuroserpin polymers. While the underlying cause of FENIB is partially understood, the mechanisms by which neuroserpin regulates the forementioned functions are still unknown. In vitro data suggest tPA as a target of neuroserpin. However, tPA-neuroserpin complexes have never been isolated in vivo and tPA-independent roles for neuroserpin have been described.
In this study, it was hypothesized that other targets besides tPA might exist. As a result, the three proteins including PC1/3, PC2 and Furin were investigated as likely targets. Due to the short-lived nature of complexes between neuroserpin and proteases, the YFP-protein fragment complementation assay was performed providing a method that enables detection of even weak or transient interactions between two proteins.
Results from this study revealed YFP-fragment complementation in cotransfections of neuroserpin with PC2, indicating interaction between the proteins. No interaction was observed in cotransfections of neuroserpin with PC1/3 or neuroserpin with Furin. However, a >170kDa neuroserpin-complex by Western blot analysis was observed when cotransfecting neuroserpin and Furin. This strongly suggests that this complex consists of neuroserpin and an unidentified protein, which possibly is activated by Furin. To further analyze the in vivo relationship between neuroserpin and PC2, the protein expression was quantified in pituitary and whole brain homogenates of neuroserpin knock-out and wild-type animals. This showed a tendency toward an inverse correlation between neuroserpin and PC2 protein levels. Follow up research will help to disclose the molecular mechanisms and further understand the relevance of these observations.