Endogenous Serine Protease Inhibitor Research Articles

The Granule Pathway of Programmed Cell Death

The exocytosis of death-inducing granzymes stored in the granules of cytotoxic lymphocytes allows the immune system to rapidly eliminate intracellular pathogens and transformed cells. The membrane-disrupting protein perforin allows the entry of granzymes into a cell, where they induce apoptosis by cleaving target substrates in the cytoplasm and nucleus. Granzymes kill cells in a variety of ways. Recent work has demonstrated that granzymes induce mitochondrial dysfunction through caspase and caspase-independent pathways and destroy DNA and the integrity of the nucleus. Cytotoxic lymphocytes are susceptible to self-inflicted damage. Mice and humans defective in perforin and granzymes point to a role for self-inflicted damage in downregulating lymphocyte responses. Given the propensity for the granule pathway to inflict cellular damage, cytotoxic lymphocytes have developed a variety of mechanisms to protect themselves. In this regard, endogenous serine protease inhibitors have been suggested to protect cytotoxic lymphocytes from granzyme B. It would appear that certain viruses and possibly even tumor cells also use the same mechanism to escape destruction from the exocytosis pathway of programmed cell death.

Functional effects of the inhibition of the cysteine protease activity of the major house dust mite allergen Der p 1 by a novel peptide-based inhibitor.

The house dust mite (HDM) Dermatophagoides pteronyssinus is an important source of allergens, which can cause allergic conditions. The cysteine protease activity of Der p 1 may enhance the potency of this major mite allergen through cleavage of CD23 and CD25 from the surface of immune cells, IgE independent mast cell activation, increases in epithelial cell permeability and inactivation of an endogenous serine protease inhibitor. Inhibition of the enzymatic activity of Der p 1 may therefore be of therapeutic benefit. To examine the activity of PTL11028, a newly developed Der p 1 inhibitor, in a range of assays that directly or indirectly measure Der p 1 protease activity and to compare its activity to endogenous cysteine protease inhibitors. The proteolytic activities of purified Der p 1 or HDM extract and inhibitory properties of PTL11028 were examined through cleavage of an artificial peptidyl substrate, cleavage of CD23 from human B cells and permeability studies on primary human bronchial epithelial cells. PTL11028 is a highly potent and specific Der p 1 inhibitor, being effective against both purified protease and Der p 1 within HDM extract. PTL11028 can completely inhibit Der p 1-mediated CD23 cleavage from human B cells and also reduces HDM-induced human bronchial epithelial cell permeability by 50%. Der p 1 is potently inhibited by cystatin A and to a lesser extent by cystatins C and E/M. PTL11028 is a highly potent and selective irreversible inhibitor of the cysteine protease activity of Der p 1, an activity that may be modulated in vivo by some human cystatins. PTL11028 prevents the Der p 1-mediated cleavage of CD23 from human B cells and significantly reduces HDM-induced permeabilization of the epithelial barrier. PTL11028 is an important tool to examine the biological effects of Der p 1 in a range of in vitro and in vivo model systems.

Induction of colligin may attenuate brain edema following intracerebral hemorrhage.

Brain edema plays an important role in the secondary brain injury following intracerebral hemorrhage (ICH). Edema formation after ICH has been linked to thrombin toxicity. Therefore, the induction of endogenous serine protease inhibitors, which inhibit thrombin prior to ICH may limit edema formation. This study examines whether injection of a low dose of thrombin upregulates such inhibitors and induces tolerance to subsequent ICH. Rats received intracerebral infusions of either one unit thrombin or saline into the right caudate nucleus. After seven days, the rats were either (A) used to examine colligin (a serine protease inhibitor) induction by Western blot analysis, immunohistochemistry and immunofluorescent double labeling, (B) to determine brain water content, or (C) they received a second injection of 50 microL blood and brain edema was determined one day later. Intracerebral infusion of thrombin caused a marked upregulation of colligin, a serine protease inhibitor, in the ipsilateral basal ganglia. Immunocytochemistry and immunofluorescent double labeling showed that colligin was induced in astrocytes. Infusion of this dose of thrombin alone did not affect brain water content but it significantly attenuated subsequent ICH-induced brain edema (79.0 +/- 0.5 vs. 81.4 +/- 0.9%, P < 0.01). Our results demonstrate that low doses of thrombin upregulate brain colligin levels and attenuate edema formation induced by ICH.

Role of Gene Overlap in the Regulation of mRNA Translation for Mitochondrial Cytochrome P-450c27/25 in the Rat

Previously published results have revealed sequence complementarity between the 5'-terminal regions of mRNAs for hepatic mitochondrial cytochrome P-450c27/ 25 (c27/25) and serine protease inhibitors (SPI) and predicted a role for this sequence overlap in both the regulation of c27/25 mRNA transcription and translation. The possibility that c27/25 mRNA forms an RNA duplex with complementary sequences of SPI mRNAs in vivo was demonstrated in the rat liver and COS-1 cells cotransfected with c27/25 and SPI2.1 plasmids. Quantitative evaluation of RNA duplex in COS-1 cells revealed that most of the c27/25 mRNA exists in duplex form when SPI2.1 mRNA was present at 5-10-fold that of c27/25 mRNA, a ratio comparable to that observed between these two RNAs in the liver. In cotransfected COS-1 cells with the same ratio of mRNAs, highly significant inhibition of the c27/25 mRNA translation (66-75%) was observed, while its transcription remained unaffected. The partial inhibition of c27/25 mRNA translation, even when most of it exists in duplex form, suggests that RNA duplex is undergoing some type of cytoplasmic processing to disengage c27/25 mRNA and make it available for translation. These results imply that abundant endogenous SPI RNAs are able to regulate the c27/25 gene expression.

Growth hormone (GH) regulation of a rat serine protease inhibitor fusion gene in cells transfected with GH receptor cDNA

The mechanism by which GH transmits a signal to the nucleus via its membrane-bound receptor is unknown. To study this process, Buffalo rat liver (BRL), rat hepatoma (FAO), human hepatoma (HepG2) and Chinese hamster ovary (CHO) cell lines were transfected with GH receptor cDNA, and stable clones expressing GH receptor mRNA and protein were selected. From previous in vivo studies it is known that GH regulates the expression of the rat hepatic serine protease inhibitor (SPI) 2.1 gene at the transcriptional level. However, in all the cell lines tested, SPI gene expression was less than 0.2% of that measured in rat liver, and GH did not affect the expression of the endogenous SPI gene in GH receptor-expressing cells. A 45 bp GH-responsive element (GHRE) has previously been defined in the SPI 2.1 gene. A construct containing six repeats of this GHRE was assembled with the thymidine kinase promoter and a chloramphenicol acetyl transferase (CAT) reporter gene. Transient transfection of this reporter gene resulted in GH stimulation of CAT activity in all GH receptor-transfected cell lines. A 33-fold induction was measured in the GH receptor-expressing BRL cells. Induction of CAT activity was observed after 8 h of GH treatment in the BRL-GHR638 cell line. Stable BRL cell lines expressing GH receptors with carboxy-terminal truncations (GHR380 and GHR454) did not show increased CAT activity on GH stimulation. This suggests that more than half of the intracellular domain of the GH receptor is required to activate transcription of the SPI 2.1 gene.(ABSTRACT TRUNCATED AT 250 WORDS)