The Phosphatidylethanolamine-binding Protein Is the Prototype of a Novel Family of Serine Protease Inhibitors

Abstract

Serine proteases are involved in many processes in the nervous system and specific inhibitors tightly control their proteolytic activity. Thrombin is thought to play a role in tissue development and homeostasis. To date, protease nexin-1 is the only known endogenous protease inhibitor that specifically interferes with thrombotic activity and is expressed in the brain. In this study, we report the detection of a novel thrombin inhibitory activity in the brain of protease nexin-1 (؊/؊) mice. Purification and subsequent analysis by tandem mass spectrometry identified this protein as the phosphatidylethanolamine-binding protein (PEBP). We demonstrate that PEBP exerts inhibitory activity against several serine proteases including thrombin, neuropsin, and chymotrypsin, whereas trypsin, tissue type plasminogen activator, and elastase are not affected. Since PEBP does not share significant homology with other serine protease inhibitors, our results define it as the prototype of a novel class of serine protease inhibitors. PEBP immunoreactivity is found on the surface of Rat-1 fibroblast cells and although its sequence contains no secretion signal, PEBP-H 6 can be purified from the conditioned medium upon recombinant expression.

A Novel Human Phosphatidylethanolamine-binding Protein Resists Tumor Necrosis Factor α-induced Apoptosis by Inhibiting Mitogen-activated Protein Kinase Pathway Activation and Phosphatidylethanolamine Externalization

“…This family is an evolutionarily conserved group found in species of flowering plants (Antirrhinum (1)), parasites (Plasmodium falciparium (2)), nematodes (Toxocara canis (3)), insects (Drosophila melanogaster (4)), and mammals, including cattle, monkeys, and humans (5). A number of functions have been suggested for the mammalian PEBP proteins, including lipid binding and inhibition of serine proteases (6). These proteins can also act as precursors for a bioactive peptide HCNP (hippocampal cholinergic neurostimulating peptide), important in hippocampus development (5).…”

A proteomic approach for the analysis of instantly released wound and immune proteins in Drosophila melanogaster hemolymph

“…Despite their widespread occurrence, in most cases the physiological role of these proteins is poorly understood. Recently, however, the mouse PEBP has been described as the prototype of a novel family of serine protease inhibitors with inhibitory activity against thrombin, neuropsin, and chymotrypsin (27). Interestingly, the human PEBP was described as a Raf-1 kinase inhibitor that modulates the mitogen-activated protein kinase-signaling cascade (28) as well as the signaling by the NF-B pathway (29).…”

“…PEBP is expressed in a wide range of tissues but was originally isolated as a cytosolic 21-to 23-kDa protein from bovine brain and binds hydrophobic ligands, in particular phosphatidylethanolamine (26). Putative PEBP homologues have been identified in a variety of organisms, including Drosophila, C. elegans, and Saccharomyces cerevisiae; the parasites Plasmodium, Onchocerca volvulus, and Toxocara canis; and the flowering plants Arabidopsis and Antirrhinum (27). Here, we show that the homologous Drosophila CG18594 protein is actually expressed.…”

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

“…The PE-binding ability of PEBP could, furthermore, involve it in the putative methylation/demethylation of PE discussed below, or, in cooperation with the membrane-binding ability of its N-and C-termini, give it a role in autophagy-related vesicle contact and fusion. Finally, given the important role of the protease Atg4 both in Atg8 activation and delipidation, the effectiveness of PEBP as a protease inhibitor 70 could bear some relevance to the regulation of autophagy.…”