Analysis of the cellular trafficking of protein C inhibor (PCI) and identification of local targets

Within this project we were able to identify and characterize a new mechanism of cellular uptake and nuclear translocation of a secreted protein (protein C inhibitor; PCI). Protein C inhibitor (PCI) is a non-specific inhibitor of proteolytic enzymes (e.g. of blood coagulation factors). PCI is a secreted, extracellular protein, which is synthesized in many organs. It inhibits its target proteases by forming stable 1:1 complexes that are enzymatically inactive. In previous immunohistochemical studies related to this project we observed that PCI might also be localized intracellularly, especially within the nuclei of leukocytes. This interesting finding, that a secreted, extracellular protein was present in the cell nucleus, prompted us to investigate the mechanism of a possible internalization of PCI by cells, and to analyze its translocation into the nucleus. By using different monoclonal antibodies we could confirm in electron microscopic studies that PCI was in fact localized within the nuclei of leukocytes and that it was associated with heterochromatin. By using labeled PCI we have shown that cells can internalize PCI and that PCI translocates to the nucleus. The internalization occurs - at least in part - directly via the phospholipid bilayer of the cell membrane. Cristallographic studies (cooperation with a group in Cambridge, UK) in fact suggest a phospholipid-binding domain within the PCI molecule. These unusual findings that a protein such as PCI can directly cross the phospholipid membrane was confirmed in studies using pure phospholipid vesicles. These phospholipid vesicles also internalized PCI. Using mutated PCI molecules we furthermore obtained data, indicating which domains within the PCI molecule are responsible for its passage through phospholipid membranes. We also performed studies concerning the translocation of PCI into the nucleus. We were able to identify domains of the PCI molecule that are necessary for nuclear translocation. As far as the biological function of intracellular/intranuclear PCI is concerned, we have obtained data suggesting that PCI interacts with proteases resulting in the cleavage of PCI. By using molecular biological techniques we have furthermore identifies several putative reaction partners of PCI.