leupeptins and acetyl-aspartyl-glutamyl-valyl-aspartal

Human mature erythrocytes have been considered as unable to undergo programmed cell death (PCD), due to their lack of mitochondria, nucleus and other organelles, and to the finding that they survive two conditions that induce PCD in vitro in all human nucleated cells, treatment with staurosporine and serum deprivation. Here we report that mature erythrocytes can undergo a rapid self-destruction process sharing several features with apoptosis, including cell shrinkage, plasma membrane microvesiculation, phosphatidylserine externalization, and leading to erythrocyte disintegration, or, in the presence of macrophages, to macrophage ingestion of dying erythrocytes. This regulated form of PCD was induced by Ca(2+) influx, and prevented by cysteine protease inhibitors that allowed erythrocyte survival in vitro and in vivo. The cysteine proteinases involved seem not to be caspases, since (i) proforms of caspase 3, while present in erythrocytes, were not activated during erythrocyte death; (ii) cytochrome c, a critical component of the apoptosome, was lacking; and (iii) cell-free assays did not detect activated effectors of nuclear apoptosis in dying erythrocytes. Our findings provide the first identification that a death program can operate in the absence of mitochondria. They indicate that mature erythrocytes share with all other mammalian cell types the capacity to self-destruct in response to environmental signals, and imply that erythrocyte survival may be modulated by therapeutic intervention.

Topics: Animals; Apoptosis; Calcium; Caspase 3; Caspases; Cysteine Endopeptidases; Death Domain Receptor Signaling Adaptor Proteins; DNA-Binding Proteins; Erythrocytes; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Macrophage Activation; Mice; Mitochondria; Models, Biological; Oligopeptides

We have previously reported the activation of procalpain mu (precursor for low-calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin.

Topics: Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Apoptosis; Aspartic Acid; Calcium-Binding Proteins; Calpain; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; fas Receptor; Humans; Jurkat Cells; Leupeptins; Oligopeptides; Poly(ADP-ribose) Polymerases; Recombinant Proteins; Tumor Necrosis Factor-alpha

Glutamate receptor stimulation reportedly activates NF-kappaB in vitro and in vivo, although underlying mechanisms remain to be elucidated. Here we evaluated the role of proteases in mediating N-methyl-D-aspartate (NMDA) receptor agonist-induced NF-kappaB activation and apoptosis in rat striatum. The intrastriatal infusion of quinolinic acid (QA, 60 nmol) had no effect on levels of NF-kappaB family proteins, including p65, p50, p52, c-Rel and Rel B. In contrast, QA decreased IkappaB-alpha protein levels by 60% (P<0. 05); other members of the IkappaB family, including IkappaB-beta, IkappaB-gamma, IkappaB-epsilon and Bcl-3, were not altered. The QA-stimulated degradation of IkappaB-alpha was completely blocked by the NMDA receptor antagonist MK-801. QA-induced IkappaB-alpha degradation and NF-kappaB activation were not affected by the proteasome inhibitor MG-132 (1-4 microg). On the other hand, the caspase-3 inhibitor Ac-DEVD.CHO (2-8 microgram) blocked QA-induced IkappaB-alpha degradation in a dose-dependent manner (P<0.05). Ac-DEVD.CHO (4 microgram) also substantially reduced QA-induced NF-kappaB activation (P<0.05), but had no effect on QA-induced AP-1 activation. Furthermore, Ac-DEVD.CHO, but not MG-132, dose-dependently attenuated QA-induced internucleosomal DNA fragmentation. These findings suggest that NF-kappaB activation by NMDA receptor stimulation involves IkappaB-alpha degradation by a caspase-3-like cysteine protease dependent mechanism. Caspase-3 thus appears to contribute to the excitotoxin-induced apoptosis in rat striatal neurons occurring at least partially as a consequence of NF-kappaB activation.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Corpus Striatum; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Fragmentation; DNA-Binding Proteins; Enzyme Activation; I-kappa B Proteins; Leupeptins; Male; Multienzyme Complexes; Neurons; NF-kappa B; NF-KappaB Inhibitor alpha; Nucleosomes; Oligopeptides; Proteasome Endopeptidase Complex; Quinolinic Acid; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Transcription Factor AP-1

Involvement of proteases has been postulated in several neurodegenerative processes. Accordingly, protease inhibition has been proposed as a potential therapeutic tool to limit damage in some neuropathological states. The timed turn-over of proteins is, however, an essential biochemical process and its prolonged block may be dangerous to the cell. We report here data on toxicity consequent to 24-h exposure of cerebellar granule neurons in culture to inhibitors of different classes of proteases. Inhibition of calpains (calcium-activated cysteine proteases) resulted in dose-dependent neuronal death which largely occurred through apoptotic process. Leupeptin, an inhibitor acting on a broad spectrum of cellular serine proteases, was less toxic but resulted in definite morphological alteration of the cells. On the contrary, inhibitors of caspases, proteases belonging to the ICE (interleukin 1-beta converting enzyme) family, did not apparently damage granule neurons upon exposure for 24 h to high concentrations (up to 200 microM) of two inhibitors specific for ICE (Ac-YAVD-CHO) and CPP-32 (Ac-DEVD-CHO), respectively. These results suggest that inhibition of proteases that are activated by stressful stimuli but are not essential for the normal functioning of healthy cells, as it is likely the case for caspases, may not be harmful to neurons. Instead, the potential risks and side effects of prolonged inhibition of proteases such as calpains, that regulate the disposal and the turn-over of key cellular proteins, should be carefully tested in the assessment of possible neuroprotective roles.

Topics: Animals; Apoptosis; Cells, Cultured; Cerebellum; Cysteine Proteinase Inhibitors; Dipeptides; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Leupeptins; Neurons; Oligopeptides; Protease Inhibitors; Rats; Rats, Wistar