Apoptosis or programmed cell death is the major mechanism used by multicellular organisms to remove infected, excessive and potentially dangerous cells. Cysteine proteases from the caspase family ...play a crucial role in the process. However, there is increasing evidence that lysosomal proteases are also involved in apoptosis. In this review various lysosomal proteases and their potential contribution to propagation of apoptosis are discussed.
We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group ...efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 microM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 microM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.
A mutant form of ammodytoxin A, a neurotoxic phospholipase A(2) from the venom of the long nosed viper Vipera ammodytes ammodytes, was prepared by site-directed mutagenesis, conjugated to a nanogold ...particle and inoculated into the antero-lateral aspect of one hind limb of female mice. Eight hours later the mice were killed, the soleus muscles of both ipsi- and contra-lateral hind limbs were removed, exposed to a silver enhancing medium and then prepared for transmission electron microscopy. Silver-enhanced particles were subsequently found concentrated in the peri-synaptic area, particularly within the synaptic gutter and the deep synaptic folds, and in many cases had been taken up into the cytoplasm of the terminal boutons of the motor axon. The results suggest that the presynaptic neurotoxicity of snake venom phospholipases A(2) involves several components of the neuromuscular apparatus, including intracellular organelles of the motor nerve terminal.
The role of the N-terminal segment 1-33 of equinatoxin II, a 20 kDa pore-forming protein from the sea anemone Actinia equina, was studied by N-truncation mutagenesis. A part of this segment was ...classified as being amphiphilic and membrane seeking. Wild-type equinatoxin II and its mutants lacking 5, 10 and 33 amino acid residues, respectively, were produced in Escherichia coli using T7 RNA polymerase-based expression vector. Soluble recombinant proteins were isolated from bacterial lysates and assayed for their inhibition by sphingomyelin, binding to red blood cells and hemolytic activity. The N-terminal deletion of 33 amino acids resulted in an insoluble protein, while mutants lacking 5 and 10 residues expressed increased relative avidity for sphingomyelin and red blood cell membranes. Their specific hemolytic activity was decreased, however, with increasing truncation. The results suggest that the N-terminus, which has been found to be conserved in sea anemone pore-forming toxins, contributes to the solubility of the equinatoxin II, but it is not essential for binding to lipid membranes. It is very likely that the N-terminus play a role in the formation of functional pores.
An important group of toxins, whose action at the molecular level is still a matter of debate, is secreted phospholipases A sub(2) (sPLA sub(2)s) endowed with presynaptic or beta -neurotoxicity. The ...current belief is that these beta -neurotoxins ( beta -ntxs) exert their toxicity primarily due to their extracellular enzymatic action on the plasma membrane of motoneurons at the neuromuscular junction. However, the discovery of several extra- and intracellular proteins, with high binding affinity for snake venom beta -ntxs, has raised the question as to whether this explanation is adequate to account for all the observed phenomena in the process of presynaptic toxicity. The purpose of this review is to critically examine the various published studies, including the most recent results on internalization of a beta -ntx into motor nerve terminals, in order to contribute to a better understanding of the molecular mechanism of beta -neurotoxicity. As a result, we propose that presynaptic neurotoxicity of sPLA sub(2)s is a result of both extra- and intracellular actions of beta -ntxs, involving enzymatic activity as well as interaction of the toxins with intracellular proteins affecting the cycling of synaptic vesicles in the axon terminals of vertebrate motoneurons.
Ammodytin L (AtnL) is a Ser-49 secretory phospholipase A sub(2) (sPLA sub(2)) homologue with myotoxic activity. By analogy to the Lys-49 sPLA sub(2) myotoxins, AtnL has been predicted to be ...enzymatically inactive due to the absence of the conserved Asp-49 that participates in coordination of the Ca super(2+) cofactor. By substituting Ser-49 and three other residues in the Ca super(2+)-binding loop of AtnL, we obtained the first two enzymatically active mutants of Lys-49/Ser-49 sPLA sub(2) homologues. The mutants LW and LV, which differed only by the presence of Trp and Val at position 31, respectively, efficiently hydrolyzed phospholipid vesicles, while recombinant AtnL displayed no activity. In contrast to AtnL but similarly to ammodytoxin A (AtxA), a homologous neurotoxic sPLA sub(2), both mutants exhibited catalysis-dependent membrane-damaging ability, involving vesicle contents leakage and fusion. However, LW and LV also exhibited the potent, Ca super(2+)-independent disruption of vesicle integrity characteristic of AtnL, but not of AtxA, in which leakage of the contents is not associated with membrane fusion. Although LV and, especially, LW have the advantage over AtnL of being able to act in both Ca super(2+)-independent and Ca super(2+)-dependent modes, and display higher cytotoxicity and higher lethal potency, they have a lower Ca super(2+)-independent membrane-damaging potency and display reduced specificity in targeting muscle fibers in vitro. Our results indicate that, in evolution, Lys-49 and Ser-49 sPLA sub(2) myotoxins have lost their Ca super(2+)-binding ability and enzymatic activity through subtle changes in the Ca super(2+)-binding network without affecting the rest of the catalytic machinery, thereby optimizing their Ca super(2+)-independent membrane-damaging ability and myotoxic activity.
A mutant form of ammodytoxin A, a neurotoxic phospholipase A sub(2) from the venom of the long nosed viper Vipera ammodytes ammodytes, was prepared by site-directed mutagenesis, conjugated to a ...nanogold particle and inoculated into the antero-lateral aspect of one hind limb of female mice. Eight hours later the mice were killed, the soleus muscles of both ipsi- and contra-lateral hind limbs were removed, exposed to a silver enhancing medium and then prepared for transmission electron microscopy. Silver-enhanced particles were subsequently found concentrated in the peri-synaptic area, particularly within the synaptic gutter and the deep synaptic folds, and in many cases had been taken up into the cytoplasm of the terminal boutons of the motor axon. The results suggest that the presynaptic neurotoxicity of snake venom phospholipases A sub(2) involves several components of the neuromuscular apparatus, including intracellular organelles of the motor nerve terminal.
Ammodytoxin A (AtxA) from the venom of Vipera ammodytes ammodytes belongs to group IIA secreted phospholipase A2 (sPLA2), for which the major pathologic activity is presynaptic neurotoxicity. We show ...here that this toxin also affects hemostasis because it exhibits strong anticoagulant activity. AtxA binds directly to human coagulation factor Xa (FXa) with Kdapp of 32 nM, thus inhibiting the activity of the prothrombinase complex with an IC50 of 20 nM. To map the FXa-interaction site on AtxA, various mutants of AtxA produced by site-directed mutagenesis and expressed in Escherichia coli were tested in the study. In surface plasmon resonance (SPR) measurements, with FXa covalently attached to the sensor chip, we show that the FXa-binding site on AtxA includes several basic amino acid residues at the C-terminal and beta-wing regions of the molecule. Applying an in vitro biological test for inhibition of prothrombinase activity, we further demonstrate that the same residues are also very important for the anticoagulant activity of AtxA. We conclude that the anticoagulant site of AtxA is located in the C-terminal and beta-wing regions of this phospholipase A2. Synthetic peptides comprising residues of the deduced anticoagulant site of AtxA provide a basis to synthesize novel anticoagulant drugs.
Recombinant lamb chymosin (RLC) was prepared and tested for its potential use in cheese production. The milk clotting activity and proteolytic activity of RLC were evaluated in comparison with ...commercial recombinant calf chymosin (RCC), cow rennet (CR), and microbial coagulant (MC). RLC, RCC, and MC showed similar responses to pH, with a sharp increase of the coagulation time at pH 6.6 to 6.8 and decrease of curd firmness at the pH 6.5 to 6.6. In the case of CR, we observed two clear increases in the coagulation time and decreases in the curd firmness, at pH 6.4 to 6.5 and 6.6 to 6.8. Optimal clotting activity was obtained for RLC at 40°C, for both CR and RCC at 45°C, and for MC at 60°C. The temperature instability of RLC at temperatures above 45°C could constitute a benefit in making hard cheese varieties. The addition of CaCl2 to milk resulted in enhanced clotting activity of all coagulants, most prominently for CR. The proteolytic activity of RLC was significantly lower from that of CR but not significantly different from the activity of RCC. The lower proteolytic activity in the cheese made with RLC did not have negative effect on organoleptic properties. The overall quality of the cheese made with RLC was at least comparable to that of the cheese made with RCC, and both cheeses were better scored than the cheese made with CR.
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