Intracellular partners of PCI

Protein C inhibitor (PCI) is a secreted non-specific serine protease inhibitor and belongs to the serpin superfamily. In humans PCI is expressed in many organs and tissues and is present in many body fluids and secretions. PCI inhibits intravascular proteases such as blood coagulation factors and fibrinolytic enzymes, but also extravascular proteases such as tissue kallikrein or the sperm protease acrosin. However, the physiological role of PCI has still not been defined.We recently generated PCI-deficient mice by targeted disruption of the PCI gene. Homozygous PCI-deficient male animals were infertile due to abnormal spermatogenesis in a testis where the blood testis barrier seemed to be disrupted. Heterozygous males as well as females of all genotypes reproduced normally. Otherwise the animals appeared normal, which was not surprising, since in mice - unlike in men - PCI is almost exclusively expressed in the reproductive tract. We recently obtained data indicating that in mouse embryos PCI can be found in many organs at certain defined stages of development, suggesting that PCI plays a role in organogenesis. Recent studies of my research group revealed furthermore that in leukocytes PCI is present not only extracellularly, but also intracellularly, especially in the nucleus. As far as the origin of this intracellular PCI is concerned, we could show that PCI can be internalized by leukocytes and HL-60 leukemia cells and then translocates to the nucleus. The function of intracellular/intranuclear PCI is unclear at present as are the mechanisms of internalisation and nuclear translocation. My research group is currently analyzing these questions. Within the proposed project we want to identify and characterize intracellular reaction partners of PCI. We will focus primarily on proteins on the cell membrane, in the cytoplasma, and/or nucleus that bind PCI. We will use different technologies including yeast-two hybrid screening, co-immunoprecipitation, and cross-linking. After having identified reaction partners of PCI, we will analyze the role of thishese interaction(s) for cell functions. Results of these studies, which will be performed in parallel to our studies analyzing the mechanisms of internalization and nuclear translocation should contribute significantly to the elucidation of the function of intracellular/intranuclear PCI.

We have shown previously that the secreted extracellular protease inhibitor protein C inhibitor (PCI) can be internalized by cells by an unconventional mechanism involving the interaction with the phospholipid phosphatidylethanolamine. Furthermore, PCI can be translocated to the nucleus. Project P17337-B09 was performed to identify intracellular or cell membrane associated proteins that interact with PCI. Using different approaches (e.g. yeast-2-hybrid screening, subcellular fractionation, co-immunoprecipitation) we were able to identify several intracellular proteins putatively interacting with PCI. The interaction with one of these proteins (JFC1, synaptotagmin-like 1) with PCI was analyzed in more detail. JFC1 is a protein that has originally been described as an interaction partner of the NADPH oxidase cytosolic factor-p67phox. JFC1 binds to phospholipids and belongs to the effector molecules of the small GTPase Rab27a, which is involved in intracellular vesicular transport. By yeast-two-hybrid screening we identified JFC1 as a PCI- binding protein. We were able to confirm the interaction of PCI with JFC1 by co-immunoprecipitation experiments not only in systems where the two proteins were overexpressed in transfected cells, but also with the endogenously synthesized proteins in leukocytes as well as in prostate cells. Furthermore we have shown by laser scanning confocal immunofluorescence microscopy that intracellular PCI and JFC1 are colocalized in close vicinity of the cell membrane. Since JFC1 has initially been identified as an interaction partner of a protein of the NADPH oxidase complex, we studied the effect of overexpression of PCI and/or JFC1 on NADPH-oxidase activity in leukocytes. With both proteins, when overexpressed separately, we observed a decrease in NADPH-oxidase activity. However, there was no additional effect when both proteins were overexpressed together. Therefore further studies are needed to determine the biolocial role of the interaction of JFC1 with PCI.