Proteases and lymphocyte cytotoxic killing mechanisms

Hudig, Dorothy; Ewoldt, Gerald R.; Woodard, Susan L.

doi:10.1016/0952-7915(93)90086-8

Cited by 69 publications

(27 citation statements)

References 40 publications

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“…Granzyme inhibition has been shown to block the lytic process (34). It is possible that calreticulin could interact with a granzyme or granzyme substrate causing lytic inhibition.…”

Section: Discussionmentioning

confidence: 99%

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Perforin Lytic Activity Is Controlled by Calreticulin

Fraser

Karimi

Michalak

et al. 2000

The Journal of Immunology

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The components within cytotoxic lymphocyte granules are responsible for a significant fraction of T and NK cell-mediated death. Perforin is stored in these granules together with calreticulin. Calreticulin has long been recognized as a chaperone protein of the endoplasmic reticulum (ER) and is the only resident ER protein to be found in the cytotoxic granules. Here we implicate a role for calreticulin in killing and report that it controls osmotic lysis mediated by purified perforin. Calreticulin, at a concentration of 2.2 × 10−7 M, completely blocked perforin-mediated lysis. Inhibition was stable and held over 5 h. Recombinant calreticulin, at a concentration of 8.8 × 10−7 M, also blocked lysis, indicating the inhibition was due to calreticulin and not a copurifying protein in the native calreticulin preparations. Using calreticulin domain fragments (expressed as GST fusion proteins), we found inhibitory activity in the high-capacity calcium-binding C-domain, which does not bind perforin. The N- or P-domains, which can bind perforin, were unable to block lysis. The inhibition of lysis was independent of granzyme inactivation or the ability of calreticulin to sequester calcium. Our data indicate that calreticulin regulation of perforin-mediated lysis probably occurs without direct interaction with perforin. We propose a novel model in which calreticulin stabilizes membranes to prevent polyperforin pore formation.

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“…Granzyme inhibition has been shown to block the lytic process (34). It is possible that calreticulin could interact with a granzyme or granzyme substrate causing lytic inhibition.…”

Section: Discussionmentioning

confidence: 99%

“…First, inactivation of granzymes (chymase or tryptase) will reduce perforin-mediated lysis (34). As a chaperone protein, calreticulin could affect either granzymes or their substrates.…”

Section: Two Mechanisms By Which Calreticulin Could Affect Lysis Appementioning

confidence: 99%

Perforin Lytic Activity Is Controlled by Calreticulin

Fraser

Karimi

Michalak

et al. 2000

The Journal of Immunology

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“…28 The serine and ICH-2-like proteases remain viable candidates for protease, granzyme B is the only other eukaryotic promediating CGN apoptosis. tease with Asp-ase activity; 18 however, since granzyme B is not found in neurons, we have excluded the Role of ICE-related cysteine proteases in other models involvement of this protease in our neuronal system. of apoptosis For a control peptide we used boc-Thr-CH 2 F, which…”

Section: Features Typical Of Apoptosis Including Morphologicalmentioning

confidence: 99%

“…A biochemical hallmark for These proteases have identical cleavage specificities apoptosis is DNA cleavage by Ca 2+ /Mg 2+ -dependent after an aspartate residue in the P 1 position (Asp-ase endonucleases into oligonucleosomal-sized fragments activity); granzyme B is the only other eukaryotic pro-(multiples of 180 bp) which migrate in a ladder pattern tease known to cleave next to Asp. 18 Hence, peptide inhibitors designed to mimic the aspartate in the P 1 position of known ICE substrates also inhibit other potassium (K + ), they undergo apoptosis. 8,20 Using this Ultrastructural analysis of neuronal apoptosis Primary neurons were cultured on 15 mm Thermanox well-characterized model we show here that the novel pan-ICE family inhibitor, boc-aspartyl(OMe)-fluoroplastic coverslips (Electron Microscopy Sciences, Ft Washington, PA, USA), treated accordingly, and then methylketone (boc-Asp-CH 2 F), protects CGN against apoptotic morphology and associated DNA fragmenfixed in 5% glutaraldehyde in PBS for 1 h at room temperature, followed by an additional overnight incutation.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of the interleukin-1β converting enzyme family rescues neurons from apoptotic death

Lynch

Vasilakos²,

Raser³

et al. 1997

Mol Psychiatry

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The interleukin-1␤ converting enzyme (ICE) family of cysteine proteases has been implicated in apoptosis. This study tested the effects of a novel pan-ICE family inhibitor, bocaspartyl(OMe)-fluoromethylketone (boc-Asp-CH 2 F), against low potassium-induced apoptosis of cultured rat cerebellar granule neurons (CGN). A single application of this cell-permeant compound (20 M) inhibited apoptotic cell death up to 48 h. Classical apoptotic changes were monitored by fluorescence microscopy, DNA fragmentation and scanning electron microscopy (SEM). A control peptidic fluoromethylketone (boc-Thr-CH 2 F), and inhibitors to calpain (Ac-Leu-Leu-norleucinal), cathepsin B (Z-Phe-Ala-CH 2 F), and CPP32-like proteases (Z-DEVD-CH 2 F), failed to prevent apoptotic death. An 35 S-methionine incorporation assay verified that, unlike cycloheximide, boc-Asp-CH 2 F did not inhibit protein synthesis, hence excluding this as a rescuing mechanism. Although ICE was not detected by northern blot analysis, both CPP32 and Nedd2 expression were found to increase during apoptosis. Kinetic assays with cell extracts from boc-Asp-CH 2 F-treated neurons measured reduced rates of cleavage for DEVD-pNA and LEVD-pNA. At present, ICE-like proteases remain viable candidates for mediating neuronal death.

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“…The group of the neutral serine protease homologs stored in the azurophilic granules of the neutrophil includes cathepsin G (Salvesen et al, 1987), elastase (Takahashi et al, 1988), proteinase 3 (Bories et al, 1989;Campanelli et al, 1990b), and the enzymatically inactive azurocidin or CAP-37 (Almeida et al, 1991;Campanelli et al, 1990a;Morgan et al, 1991), which are cationic glycoproteins of similar size (25-29 kD) which have been cloned. Neutrophil serine proteinases exhibit sequence homologies between each other and with T cell proteases, human lymphocyte proteases, granzyme B, and rat mast cell proteases (Hudig et al, 1993). Genomic cloning has revealed that neutrophil elastase, proteinase 3, and azurocidin genes form a cluster of genes, located in the terminal region of the short arm of chromosome 19 and coordinately regulated in the promonocytic cell line U937 during induced terminal differentiation (Sturrock et al, 1992;Zimmer et al, 1992).…”

Section: Granule Proteinsmentioning

confidence: 99%