L-DNase II, a Molecule That Links Proteases and Endonucleases in Apoptosis, Derives from the Ubiquitous Serpin Leukocyte Elastase Inhibitor

Abstract

The most widely recognized biochemical change associated with the majority of apoptotic systems is the degradation of genomic DNA. Among the enzymes that may participate in this cleavage, the acidic cationindependent DNase II is a likely candidate since it is activated in many apoptotic cells. To better understand its role, we purified and sequenced a DNase II extracted from porcine spleen. Protein sequencing of random peptides demonstrated that this enzyme is derived from a ubiquitous serpin, the leukocyte elastase inhibitor (LEI), by an acidic-dependent posttranslational modification or by digestion with elastase. We call this novel enzyme L-DNase II. In vitro experiments with purified recombinant LEI show that the native form has no effect on purified nuclei whereas its posttranslationally activated form induces pycnosis and DNA degradation. Antibodies directed against L-DNase II showed, in different cell lines, an increased expression and a nuclear translocation of this enzyme during apoptosis. Since the appearance of the endonuclease activity results in a loss of the anti-protease properties of LEI, the transformation from LEI to L-DNase II may act as a switch of protease and nuclease pathways, each of which is activated during apoptosis.Apoptosis, or programmed cell death, is a mechanism of cell clearance in many physiological processes such as embryogenesis, metamorphosis, and tumor regression (2). Although the signals inducing apoptosis are very different, nuclear condensation, membrane blebbing, and formation of apoptotic bodies are morphological features common to all apoptotic cells. By far the most widely recognized biochemical change is the degradation of genomic DNA. The identity of the enzymes responsible for this cleavage is the subject of considerable debate. Several endonucleases have been proposed to be responsible for DNA fragmentation. Ca 2ϩ -plus Mg 2ϩ -dependent endonucleases in thymocytes, such as NUC 18/cyclophylin A (16), DNase I (21), DNase ␥ (29), and a new 97-kDa DNase (18), are examples. Mg 2ϩ -dependent, Ca 2ϩ -independent endonucleases have been implicated in human myeloid cell line apoptosis (7,8). Barry and Eastman (1) implicated DNase II, a cation-independent acidic endonuclease, as the enzyme that degrades DNA in apoptosis associated with intracellular acidification. We have shown in our laboratory the involvement of DNase II in nuclear degradation in terminally differentiating lens fiber cells (32).To date, our knowledge of the molecular structure of DNase II is very limited. The enzymatic properties of DNase II from different tissues and animals were found to be very similar, but its physical and chemical properties showed high variability. For instance, the molecular mass of mammalian DNase II ranges between 26 and 45 kDa. The reasons for this variability remain unknown (15).To better understand the biology of DNase II, the knowledge of its protein sequence seemed to be a mandatory step. In this study, we showed that this ubiquitous L-DNase II arises from leukocyte elastase...

Regulation of DNaseY activity by actinin-α4 during apoptosis

“…For example, some of the enzymes translocate to the nucleus upon receiving an apoptotic signal, that is, endoG from mitochondria and DNaseI from the rough endoplasmic reticulum, 11,12 whereas others, namely L-DNaseII and Helicard, are activated by proteolytic cleavage of their precursors. 13,14 Activation of CAD occurs as a result of proteolysis of its inhibitor ICAD. In proliferating cells, ICAD functions as a CAD-specific chaperone, but upon receiving an apoptotic stimulus, caspases cleave it permitting CAD to degrade chromosomal DNA.…”

Functional Characterization of DNase X, a Novel Endonuclease Expressed in Muscle Cells

“…Important mechanisms include the proteolysis of an inhibitor as in the case of CAD/ICAD (15), or an acidic pH-dependent 1 Abbreviations: CAD, caspase-activated DNase; DAPI, 4,6-diamidine-2-phenylindole; DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin; DTT, dithiothreitol; GFP, green fluorescent protein; ICAD, inhibitor of CAD; PARP, poly(ADP-ribose)polymerase; PBS, phosphate-buffered saline; PMSF, phenylmethanesulfonyl fluoride; TBS, Tris-buffered saline; zVAD, benzyloxycarbonyl-Val-Ala-Asp-(OMe)fluoro-methylketone.…”

Structural requirements of human DNase IIalpha for formation of the active enzyme: the role of the signal peptide, N-glycosylation, and disulphide bridging

“…Acid endonucleases were originally reported at a variety of sizes [2,[11][12][13][14], and subsequent identification of distinct enzymes has suggested that several unrelated enzymes appear to contribute to the acidendonuclease activity found in mammalian tissues. These include DNase II, L-DNase II, Xib, DNase α and DNase β [1,[15][16][17][18][19]. In addition to these unrelated enzymes, a new homologue of human DNase II has been recently cloned, named DNase IIβ (hence the original enzyme is now termed DNase IIα) [20].…”