Hypothesis: Cytotoxic Lymphocyte Granule Serine Proteases Activate Target Cell Endonucleases to Trigger Apoptosis

Smyth, Mark J.; Browne, Kylie A.; Thia, Kevin; Apostolidis, Vicki A.; Kershaw, Michael H.; Trapani, Joseph A.

doi:10.1111/j.1440-1681.1994.tb02438.x

Cited by 21 publications

(15 citation statements)

References 22 publications

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“…Alternatively, many of the proteases/nucleases may actually have a downstream function to fully degrade and recycle macromolecules following cell death. Also, it has been suggested that the means by which proteases could contribute to chromatin cleavage would be to activate nucleases themselves, freeing them from an inhibitory complex or conformation (19,52).…”

Section: Discussionmentioning

confidence: 99%

Purification, Characterization, and Role of Nucleases and Serine Proteases in Streptomyces Differentiation

Nicieza

Huergo

Connolly

et al. 1999

Journal of Biological Chemistry

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Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described in Streptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5-phosphate mononucleotides as the predominant products. Both proteins require Mg 2؉ , and the additional presence of Ca 2؉ notably stimulates their activities. The two nucleases are inhibited by Zn 2؉ and aurin tricarboxylic acid. The 18-kDa nuclease from Streptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by aminoterminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolyl cistrans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and "native" cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited by N ␣ -ptosyl-L-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells.The actinomycetes are a large group of filamentous bacteria that are adapted for growth in soil by forming a ramifying network, called a mycelium. Within this group the predominant isolates belong to the genus Streptomyces, which produce a well developed branched mycelium on agar plates, resulting in a compact colony. In the vegetative phase, the filaments often lack cross-walls (substrate mycelium) and thus have several copies of the chromosome. When the colony ages, a characteristic aerial mycelium is formed, in response to unknown signals involving nutrient limitation, which subsequently fragment and/or sporulate by the synchronous formation of crosswalls in the multinucleate sporophores followed by separation of the individual cells directly into spores. This is similar to the growth and differentiation of fungi, and from the morphological and metabolic points of view, Streptomyces can be considered as boundary organisms (1). Coincident with the morphological differentiation, the streptomycetes produce numerous compounds (secondary metabolites) within which antibiotics are of commercial relevance. As corresponds to their habitat, these bacteria are nutritionally quite versatile, and most produce extracellular hydrolytic enzymes that permit the utilization of polysaccharides, proteins, fats, and other substrat...

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Section: Discussionmentioning

confidence: 99%

Purification, Characterization, and Role of Nucleases and Serine Proteases in Streptomyces Differentiation

Nicieza

Huergo

Connolly

et al. 1999

Journal of Biological Chemistry

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“…Lamin A, lamin B, poly(ADPribose) polymerase, and topoisomerases are all targets of proteolytic degradation during apoptosis in many systems (39,47,48). As is the case for protein B23, nucleolin is another nuclear matrix protein that is localized to the nucleolus and involved in rRNA synthesis and has been shown to be a preferential target of serine proteases during programmed death induced in target cells by cytotoxic lymphocytes (49). Although the nature of the proteases responsible for degradation of these nuclear matrix proteins, including protein B23, is still unknown, a Ca 2ϩ -dependent protease exists in the nuclear lamina (39) and may be involved in this process (40).…”

Section: Fig 2 Effect Of Androgen Deprivation On Steady-state Level Ofmentioning

confidence: 99%

Role of Protein Phosphorylation in Post-translational Regulation of Protein B23 during Programmed Cell Death in the Prostate Gland

Tawfic

Olson

Ahmed

1995

Journal of Biological Chemistry

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Protein B23 is a nucleolar and nuclear matrix-associated phosphoprotein that is involved in ribosome synthesis. Its expression and phosphorylation in rat ventral prostate, an androgen target organ, are profoundly influenced by androgens. Induction of programmed cell death (apoptosis) in the prostatic epithelium by androgen deprivation in the animal induces an early decline in protein B23 in the absence of a corresponding loss of protein B23 mRNA. We have now demonstrated that prostatic nuclei retain the ability to transcribe the B23 mRNA and that a significant amount of this mRNA persists even after 7 days of androgen deprivation when >80% of the prostatic epithelial cells have undergone apoptosis. The B23 mRNA from these nuclei is also translatable in vitro. However, the majority of the B23 mRNA is associated with free and short-stretch polysomes, which may account for the castration-induced decline in synthesis of protein B23 in vivo. In addition, the mechanism of down-regulation of protein B23 in apoptotic prostatic cells appears to relate to two coordinate signals, which include loss of phosphorylation of the protein as well as the expression of a protease active toward dephosphorylated protein B23, under these conditions.Programmed cell death or apoptosis has been described in diverse biological systems mediated by a variety of signals (1-3), and the response of the prostatic glandular epithelium to androgen withdrawal is one of the frequently studied models (3, 4). Androgen withdrawal via orchiectomy in the rat induces an energy-dependent cascade of biochemical and morphological changes that lead to the death of 80% of the secretory epithelium between days 2 and 6 (4). Morphologically, the nucleolus dissolves (5), and the chromatin is condensed and fragmented to form the membrane-bound apoptotic bodies that appear in appreciable numbers after 2 days of androgen deprivation (6). Biochemically, there is an increase in intracellular calcium and enhancement of calcium/magnesium-dependent endonuclease activity that reaches its maximum 4 -5 days after androgen withdrawal (7). Apoptosis in the prostate is associated with modulation of gene expression so that the expression of some genes is repressed and that of others is enhanced. Among the latter are c-fos, c-myc, heat shock proteins (8), glutathione S-transferase (9), and TRPM-2/sulfated glycoproteins (10). On the other hand, the synthesis of ribosomes, especially their assembly into polysomes, markedly declines after androgen deprivation (11,12). Also, prostatic ribosomes from animals treated with 5␣-dihydrotestosterone support a significantly higher incorporation of radiolabeled amino acids into proteins than do ribosomes isolated from castrated animal controls (11).We have been interested in the mechanisms underlying the decline in prostatic rRNA synthesis and assembly after androgen deprivation. Protein B23, a conserved phosphoprotein that is localized to the granular and fibrillar regions of the nucleolus where rRNA synthesis and assembly take place (13, 1...

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“…Inhibition of granzyme proteolytic activity blocks perforin-dependent pore formation in both hemolytic assays and NK cell-mediated assays (12). Granzymes also initiate the procaspase apoptotic cascade leading to a second type of granule-initiated target cell death, apoptosis (13,14). After granule-mediated necrosis or apoptosis, the killer cell is unharmed, suggesting that a protective mechanism remains to be discovered.…”

Section: Perforin Lytic Activity Is Controlled By Calreticulinmentioning

confidence: 99%

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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Hypothesis: Cytotoxic Lymphocyte Granule Serine Proteases Activate Target Cell Endonucleases to Trigger Apoptosis

Cited by 21 publications

References 22 publications

Purification, Characterization, and Role of Nucleases and Serine Proteases in Streptomyces Differentiation

Purification, Characterization, and Role of Nucleases and Serine Proteases in Streptomyces Differentiation

Role of Protein Phosphorylation in Post-translational Regulation of Protein B23 during Programmed Cell Death in the Prostate Gland

Perforin Lytic Activity Is Controlled by Calreticulin

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