Membrane associated proteases and protein C inhibitor

Protein C inhibitor (PCI) is a glycoprotein, which inhibits serine proteases by forming stable 1:1 complexes. PCI is synthesized in many organs and tissues and is present in many body fluids. It has broad protease reactivity, and its activity towards most proteases is stimulated by binding to glycosaminoglycans (e.g. heparin). Recently the group of the applicant has shown that PCI also binds to certain anionic and/or oxidized phospholipids and that these phospholipids stimulate its inhibitory activity (Malleier et al., Blood 2007). So far only the interactions of PCI with target proteases present in body fluids (e.g. blood coagulation and fibrinolytic enzymes, members of the tissue kallikrein family) have been studied. However, serine proteases (or their inactive precursors) are present not only in body fluids, but also occur as cell membrane associated proteins. They can either be transmembrane proteins or anchored via a GPI-anchor. Membrane associated serine proteases seem to play a role in cell proliferation and migration and therefore also in tumor growth and metastasis. At present it is unknown to which extent these membrane anchored serine proteases are inhibited by serine protease inhibitors. We hypothesize that PCI could play a role for the regulation of these proteases, since PCI has affinity for phospholipids and glycosaminoglycans and since it is widely distributed in the organism. It is the aim of this project to analyze the interaction of PCI with membrane associated serine proteases and to determine the effect of phospholipids and glycosaminoglycans on this interaction. We will analyze the interaction of PCI with the protease testisin in detail, since preliminary in vitro data suggest that this protease is a PCI target, and since PCI and testisin are colocalized in male germ cells and in some tumor cells. In the second part of the project we want to identify new membrane associated target proteases of PCI and to study their interaction with PCI as well as their colocalization in subcellular compartments. We will use different (tumor) cell lines that express PCI as well as cells that are exposed to PCI (e.g. peripheral blood leukocytes). From these studies we expect information on the role of PCI for the regulation of local, cell associated proteolytic activity.

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 phosphatidylethanolamine and further translocated to the nucleus. Aim of the project was to find new membrane associated and intracellular proteins that interact with PCI. To identify interaction partners of PCI subcellular fractionation and co- immunoprecipitation were performed. We were able to find new membrane associated target proteases: enteropeptidase, a type II transmembrane serine protease and testisin, a GPI- anchored serine protease. Furthermore we have obtained results showing that PCI is also interacting with intracellular proteins. CSN6 and CSN5, subunits of COP9 signalosome, and a nuclear isoform of the lysosomal cathepsin L were co-localized with PCI in subcellular fractions of peripheral blood leukocytes. In Western blots PCI antigen was detected in the nucleus of lymphocytes, whereas in the nucleus of Jurkat lymphoma cells there was no detectable PCI antigen, consistent with published data showing that the absence of Protein C inhibitor (PCI) is associated with malignant behavior. It is known that PCI regulates the activity of secreted cathepsin L of cancer cells. Therefore we wanted to investigate if PCI is also an inhibitor of nuclear isoform of lysosomal cathepsin L. Jurkat T lymphoma cells were incubated with PCI. PCI was internalized and could be co-localized with nuclear cathepsin L in the chromatin. By inhibiting nuclear cathepsin L PCI prevented the proteolytic processing of histone H3, one of the core histones closely associated with the DNA. We could furthermore show for the first time that proteolytic processing of histone H3 by cathepsin L inhibits a chemical modification of histone H4. Absence of this modification is a hallmark for cancer cells. Internalized PCI could rescue that modification on histone H4. These histone modifications are responsible for the structure of chromatin and play important roles in the regulation of gene expression. As a regulator of nuclear cathepsin L PCI can prevent epigenetic modifications typical for cancer, internalized PCI is therefore a putative tumor suppressor.