Proteasi nexina-1: produzione e caratterizzazione funzionale della proteina ricombinante, e studi strutturali mediante "molecular modelling".

Protease Nexin-1 (PN-1) is a member of the serpin superfamily which mainly inhibits Thrombin; this inhibition is enhanced by the interaction with heparin. This protein is involved in embryogenesis of the nervous system and of the sexual organs and probably exherts a role in a variety of pathologies such as Alzheimer disease and scleroderma. Even though this protein can be obtained by the media of different cell lines and different systems to obtain recombinant PN-1 have been developed, the yields are in general rather low. For this reason in our lab we have tried to develop a method to produce recombinant PN-1 and obtain higher yields compared to the ones previously reported to be able to perform a structural study of this protein. Two different expression systems have been used: hamster BHK cells and E. coli. Both systems have given satisfactory yields but the one in E. coli,because of slightly higher yields of expressed PN-1 and because of the easier preparation resulted as best. At the end of the process of purification the total yield was of 0.2-0.15 mg/L of bacterial colture. Recombinant PN-1 has been characterized for its inhibitory and cellular activities. Recombinant PN-1 correctly inhibits Thrombin: this interaction is, as for the native protein, enhanced by heparin. Furthermore Thrombin mutants, previously produced in our lab, correctly recognise and cleave PN-1 only if still enzimatically active. We have also characterized the binding of PN-1 to heparin determining the equilibrium interaction constant (Kd=31nM). The cellular activity of PN-1 has been tested by analysing its ability to cause stellation in neural cells. We used Nb2A mouse nuroblastoma cells, and a recombinant PN-1 concentration of 50 nM has been able to cause complete stellation. We have also employed the technique of molecular modelling to perform a structural analysis of PN-1 using two serpins, PAI-1 and ATIII as models. First of all we localised the heparin binding site on the structure helix D and the site responsable for Thrombin-PN-1 complexes clearance on the loop connecting helices A and B. We also showed that presumably the interaction with heparin does not cause, for PN-1, the same structural activation of ATIII. These results show how it has been possible to develop a method to produce a recombinant PN-1 and that this protein is active both as an inhibitor and on cells. This protein will be used to confirm and widen the structural data obtained by molecular modelling. The recombinant PN-1 so obtained could also be used to invetigate the phisiological and pathological activities that have been connected to this serpin in the past few years.