Showing papers on "Pore forming protein published in 1996"

Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O.

Abstract: Plasmodium falciparum is an intracellular parasite of human red blood cells (RBCs). Like many other intracellular parasites, P. falciparum resides and develops within a parasitophorous vacuole which is bound by a membrane that separates the host cell cytoplasm from the parasite surface. Some parasite proteins are secreted into the vacuolar space and others are secreted, by an as yet poorly defined pathway, into the RBC cytosol. The transport of proteins from the parasite has been followed mainly using morphological methods. In search of an experimental system that would allow (i) dissection of the individual steps involved in transport from the parasite surface into the RBC cytosol, and (ii) an assessment of the molecular requirements for the process at the erythrocytic side of the vacuolar membrane, we permeabilized infected RBCs with the pore-forming protein streptolysin O using conditions which left the vacuole intact. The distribution of two parasite proteins which served as markers for the vacuolar space and the RBC cytosol respectively was analysed morphologically and biochemically. In permeabilized RBCs the two marker proteins were sorted to the same compartments as in intact RBCs. The protein which was destined for the RBC cytosol traversed the vacuolar space before it was translocated across the vacuolar membrane. Protein transport could be arrested in the vacuole by removing the RBC cytosol. Translocation across the vacuolar membrane required ATP and a protein source at the erythrocytic face of the membrane, but it was independent of the intracellular ionic milieu of the RBC.

Molecular executors of cell death--differential intrarenal expression of Fas ligand, Fas, granzyme B, and perforin during acute and/or chronic rejection of human renal allografts.

Abstract: Two distinct cytolytic pathways have been characterized : one in which the interaction between the Fas antigen and its ligand results in apoptosis, and another in which the pore forming protein perforin and the serine protease granzyme B contribute to DNA fragmentation and cell death. We investigated intrarenal expression of these molecular executors of cell death in light of the potential participation of cytolytically active cellular elements in the antiallograft repertory. Reverse transcriptase-polymerase chain reaction was used to identify intrarenal expression of Fas antigen, Fas ligand, granzyme B and perforin in eighty human renal allograft biopsies; mRNA display was correlated with the Banff histological diagnosis of renal allografts. Our studies demonstrate that: (1) intrarenal expression of Fas ligand mRNA and of granzyme B mRNA are correlates of acute but not chronic rejection ; (2) Fas ligand mRNA is not detectable in allografts in the absence of rejection; (3) intrarenal coexpression of members of each lytic pathway (Fas ligand and Fas, granzyme B, and perforin) and that of both pathways (e.g., Fas ligand and granzyme B) are correlates of acute rejection; and (4) a direct correlation exists between the histological severity of acute rejection and intrarenal coexpression of mRNA encoding Fas ligand, Fas, granzyme B, and perforin. Our studies identify, for the first time, the differential expression of the two major lytic pathways in acute and chronic allograft rejection and suggest that specific therapy directed at the cytotoxic attack molecules might be efficacious in the prevention and/or treatment of acute rejection.

Granzymes: a variety of serine protease specificities encoded by genetically distinct subfamilies.

Abstract: Granzymes are a family of granule serine proteases found specifically in the cytotoxic granules of cytotoxic T lymphocytes and natural killer cells. Granzymes have features that are strongly conserved including: consensus sequences at their N-termini and around the three catalytic residues, activation from zymogenic forms, and conserved disulphide bridges. However, there is good genetic evidence to suggest that three distinct subfamilies of granzymes have coevolved. These subfamilies are most strikingly depicted by their distinct chromosomal loci and gene organization, dividing the granzyme family into subfamilies of the following: tryptases (human chromosome 5); chymotrypsin-like proteases (human chromosome 14); and a Metase amongst a cluster of elastase-like proteases (human chromosome 19). Modeling and mutational analysis has revealed that each subfamily of granzymes displays special sequence and structural features and a proteolytic specificity determined by subtle modifications to substrate binding pocket residues. It now remains of great interest to determine whether these subfamilies also possess distinct biological functions. Granzyme B has been shown to play an important role in lymphocyte-mediated target cell apoptosis and the tryptase, granzyme A, has been demonstrated to regulate the clearance of some pox virus infections. The future creation of other granzyme gene knockout mice should elucidate whether other chymotrypsin-like granzymes (C-H) also contribute to target cell apoptosis and whether the third subfamily member, natural killer cell-specific Metase, has a distinct biological function.

Cytolytic activity in the genus Leishmania: involvement of a putative pore-forming protein.

Abstract: We describe here that parasites of the genusLeishmaniacontain a cytolytic activity which acts optimally at pH 5.0 to 5.5 and at 37&C in vitro. Of the four species examined, Leishmania (Leishmania) amazonensis and Leishmania(Leishmania)majorpresented considerable hemolytic activity, whereasLeishmania(Viannia)panamensisandLeishmania(Viannia)guyanensisshowed little and no hemolytic activity, respectively. The cytolytic factor of L. amazonensis promastigotes was characterized as a protein with no protease-, phospholipase-, or detergent-like activity, probably localized inside membranous vesicles. The use of osmotic protectants revealed the colloid-osmotic nature of hemolysis, which is indicative of pore formation in the membranes of target cells. This putative pore-forming protein also damaged nucleated cells, including macrophages, causing an increase in their membrane permeability with leakage of cytoplasmic proteins. Both promastigotes and amastigotes express this lytic activity, suggesting that the cytolysin may have a function in both stages of this parasite. The pH and temperature required for optimal activity indicate that it might be more effective within the mammalianhost,particularlyinsidethemacrophageparasitophorousvacuole.InpromastigotesofL.amazonensis,the expression of lytic activity seems to be regulated during their growth in vitro, being maximal at the early stationary phase.

Activation of apoptosis pathways by granzyme B.

Abstract: Cytotoxic lymphocytes induce apoptosis of target cells by degranulating and releasing the serine protease granzyme B and the pore forming protein perforin. Granzyme B is an aspartic acid protease similar to members of the interleukin 1beta converting enzyme (ICE) family. We review the evidence for the participation members of the ICE family of proteases and cdc2 kinase in granzyme B-induced apoptosis.

Cell‐free Conversion of a Ubiquitous Nuclear Protein into a Killer‐Cell‐Specific form that Binds to the Nf‐P Enhancer Element of the Mouse Perforin Gene

Abstract: Two nuclear factors, designated NF-PI and NF-P2, have been shown to bind to an enhancer 9-base motif (5'-ACAGGAAGT-3', NF-P motif) present within the 5'-flanking region of the mouse perforin gene. Our previous studies have shown that, although NF-P1 and NF-P2 differ in cell-type distribution and molecular mass, with NF-P2 being killer-cell-specific and smaller, the two factors appear to share common DNA-binding subunit(s). We have postulated that the biochemical event involved in the induction of NF-P2 could be the dissociation of a non-DNA-binding subunit from NF-P1, rendering the newly formed NF-P2 transcriptionally active. By using a cell-free system in the present study, we have demonstrated that a variety of chemical agents capable of denaturing or dissociating protein complexes, including guanidinium/HCl, detergents (SDS plus Nonidet P-40) and high-salt solutions, could convert NF-P1 into NF-P2. Unlike in intact cells, where induction of NF-P2 is restricted to killer lymphocytes, this conversion occurred in nuclear extracts derived from both cytotoxic lymphocytes and non-cytotoxic cells. Although the mechanism that restricts the induction of NF-P2 to killer- lymphocytes in vivo remains unresolved, these results support the hypothetical 'dissociation' model for the generation of NF-P2. The results also imply that the absence of perforin expression in non-cytotoxic cells may be due to the suppression of the induction of the killer-cell-specific trans-acting factor NF-P2.

Cell membrane damage in lymphocyte-mediated cytolysis: Role of lymphocyte pore-forming protein perforin

Abstract: Publisher Summary This chapter discusses the theory of cell membrane damage in lymphocyte-mediated cytolysis—role of lymphocyte pore-forming protein perforin. Conjugation with the target cell triggers degranulation by the killer cell, which then results in the release of a number of putative cytotoxins into the intercellular space. A prominent member among these toxins is the pore-forming protein A; a large number of pore-forming toxins are known to create channels through a “barrel stave” mechanism. Three discrete steps have been defined for the pore formation in this model: (1) binding of the water-soluble monomers to the membrane, (2) insertion of the monomers into the membrane, and (3) aggregation of the monomers, such as barrel staves, surrounding a central pore such that the hydrophobic side of the proteins is exposed to the membrane acyl chains and the hydrophilic side lines up the pore. As a result, stable transmembrane pores are formed that allow ions and small molecules to flow passively across the bilayer. As many of the homopolymeric channels formed by toxins are not large enough to allow the passage of cytoplasmic proteins, this model predicts that the channels would produce an ionic imbalance in the cell, which then results in colloid osmotic lysis. Perforin is likely to lyse target cells by this mechanism.