Possible Involvement of Cyclophilin B and Caspase-Activated Deoxyribonuclease in the Induction of Chromosomal DNA Degradation in TCR-Stimulated Thymocytes

Abstract

TCR engagement of immature CD4+CD8+ thymocytes induces clonal maturation (positive selection) as well as clonal deletion (negative selection) in the thymus. However, the cell death execution events of thymocytes during the negative selection process remain obscure. Using a cell-free system, we identified two different DNase activities in the cytosol of in vivo anti-TCR-stimulated murine thymocytes: one that induced chromosomal DNA fragmentation, which was inhibited by an inhibitor of caspase-activated DNase, and another that induced plasmid DNA degradation, which was not inhibited by an inhibitor of caspase-activated DNase. We purified the protein to homogeneity that induced plasmid DNA degradation from the cytosol of anti-CD3-stimulated thymocytes and found that it is identical with cyclophilin B (Cyp B), which was reported to locate in endoplasmic reticulum. Ab against Cyp B specifically inhibited the DNA degradation activity in the cytosol of anti-CD3-stimulated thymocytes. Furthermore, recombinant Cyp B induced DNA degradation of naked nuclei, but did not induce internucleosomal DNA fragmentation. Finally, we demonstrated that TCR engagement of a murine T cell line (EL4) with anti-CD3/CD28 resulted in the release of Cyp B from the microsome fraction to the cytosol/nuclear fraction. Our data strongly suggest that both active caspase-activated DNase and Cyp B may participate in the induction of chromosomal DNA degradation during cell death execution of TCR-stimulated thymocytes.

The Contribution of Apoptosis-inducing Factor, Caspase-activated DNase, and Inhibitor of Caspase-activated DNase to the Nuclear Phenotype and DNA Degradation during Apoptosis

“…One of the genes found, CYP-13, belongs to the family of cyclophilins, which have been previously involved in chromosomal DNA degradation in vertebrates. Cyclophilins show a nuclease activity distinct from the cis-transisomerase one, and their ionic requirements are similar to that described for apoptotic nucleases (29,30). Cyp C has been shown to induce 50-kbp DNA fragmentation on isolated nuclei (29), whereas Cyp A has been demonstrated to interact and cooperate with AIF in apoptosis-associated chromatinolysis (31).…”

Recombinant Cyclophilins Lack Nuclease Activity

“…For the aforementioned eukaryotic cyclophilins, the accompanying nuclease activity has been proposed as playing a central role in apoptosis. This is the case with NUC-18, a nuclease reported to be identical to rat CypA cyclophilin, involved in glucocorticoid-stimulated apoptosis of thymocytes (7) and murine CypB cyclophilin-associated nuclease, involved in TCR-stimulated apoptosis of thymocytes (8). For S. antibioticus SanCyp18 cyclophilin, nuclease activity has been related to chromosomal DNA degradation during the lysis of substrate (vegetative) mycelium (2), which precedes the emergence of the aerial (reproductive) mycelium (9).…”

“…Minor contamination of cyclophilins with these or other nucleases can lead to erroneously associating these activities with the cyclophilin proteins, as occurred in the case of SanCyp18 and LpCyp18 human recombinant cyclophilins. Other native cyclophilins described as nucleases, such as NUC18 (7), murine Cyp B (8), and SanCyp18 (9), might also conceivably be contaminated by host activities. The presence of nuclease-contaminated cyclophilin samples could equally explain some published results on the inhibition of cyclophilin-associated nuclease activity by cyclophilin antibodies (8).…”

“…Other native cyclophilins described as nucleases, such as NUC18 (7), murine Cyp B (8), and SanCyp18 (9), might also conceivably be contaminated by host activities. The presence of nuclease-contaminated cyclophilin samples could equally explain some published results on the inhibition of cyclophilin-associated nuclease activity by cyclophilin antibodies (8). In summary, it can be concluded that SanCyp18 and human and Legionella recombinant cyclophilins lack nuclease activity; consequently, other published data concerning the presence of this activity in native cyclophilins should be analogously reappraised.…”

“…Some reports on human recombinant CypA, CypB, and CypC cyclophilins (6), murine CypB cyclophilin (8), Legionella pneumophila LpCyp18 cyclophilin (12), and Streptomyces antibioticus-SanCyp18 cyclophilin (9) describe associated Ca 2ϩ / Mg 2ϩ nuclease-dependent activity. For the aforementioned eukaryotic cyclophilins, the accompanying nuclease activity has been proposed as playing a central role in apoptosis.…”