Conformational modification of serpins transforms leukocyte elastase inhibitor into an endonuclease involved in apoptosis

Abstract

The best-characterized biochemical feature of apoptosis is degradation of genomic DNA into oligonucleosomes. The endonuclease responsible for DNA degradation in caspase-dependent apoptosis is caspase-activated DNase. In caspase-independent apoptosis, different endonucleases may be activated according to the cell line and the original insult. Among the known effectors of caspase-independent cell death, L-DNase II (LEI [leukocyte elastase inhibitor]-derived DNase II) has been previously characterized by our laboratory. We have thus shown that this endonuclease derives from the serpin superfamily member LEI by posttranslational modification (A. Torriglia, P. Perani, J. Y. Brossas, E. Chaudun, J. Treton, Y. Courtois, and M. F. Counis, Mol. Cell. Biol. 18:3612-3619, 1998). In this work, we assessed the molecular mechanism involved in the change in the enzymatic activity of this molecule from an antiprotease to an endonuclease. We report that the cleavage of LEI by elastase at its reactive center loop abolishes its antiprotease activity and leads to a conformational modification that exposes an endonuclease active site and a nuclear localization signal. This represents a novel molecular mechanism for a complete functional conversion induced by changing the conformation of a serpin. We also show that this molecular transformation affects cellular fate and that both endonuclease activity and nuclear translocation of L-DNase II are needed to induce cell death.

FIG. 1.

Sequence alignment of horse, human, and pig LEI proteins. By convention, amino acid numbering refers to the PDB nomenclature of serpins that aligns all of the serpins with α1-antitrypsin. UniProtKB/Swiss-Prot entries: horse, LEI P05619; human, LEI P30740; pig, LEI P80229. The hinge region is light green, the inhibitory consensus pattern is dark green, and the P1-P1′ elastase cleavage site is blue. The RCL integrated into the main β-sheet of LEI after cleavage is in bold, and the single elastase recognition site is underlined. His 287, which is lost in human LEI and replaced with Ser, is cyan. The two cysteines of pig LEI are purple. The sequences obtained by Edman degradation are yellow (41). The bipartite NLS is red, and the endonuclease active site is orange. Red arrowheads indicate points of site-directed mutagenesis (1, main NLS mutant; 2, hinge region mutant; 3, endonuclease active-site mutant).

FIG. 2.

Antiprotease and endonuclease activities of three different LEI mutants. Site-directed mutagenesis was performed with a wild-type (WT or wt) LEI-pET 23d(+) construct and the QuikChange mutagenesis kit (Stratagene). An alanine residue of the hinge region of the RCL of LEI (AP10T mutant) and histidine 368 of the endonuclease active site (H368A mutant) were changed to threonine and alanine, respectively. Recombinant proteins were produced by E. coli strain BL21/pLysS and purified with His-select cartridges (Sigma). Increasing concentrations of wild-type or mutant LEI (17.5 to 280 μg/ml) (magenta and blue lines) were incubated with elastase (0.1 μg/ml) in the presence of a 2 mM concentration of a synthetic substrate, pNaMAAPV. The resulting colored reaction was measured (DO, optical density) at 405 nm (a). As a control, elastase and its substrate were incubated alone (red). In the AP10T mutant, the antiprotease activity is strongly affected compared to that of wild-type LEI. On the contrary, this activity is not affected in the H368A mutant. DNase activity (b) was measured after overnight cleavage of LEI with equimolar quantities of elastase at 37°C. Samples (841 ng) of wild-type, AP10T, and H368A LEI were incubated with 2.5 μg of plasmid DNA for different times at 37°C in 20 mM Tris-EDTA, pH 5.5. Untreated wild-type LEI was incubated with DNA as a negative control. The AP10T mutant shows a DNase activity comparable to that of the wild type, while H368A has no endonuclease activity. No endonuclease activity could be detected for the 358stop mutant, but DNA was slowed down with incubation time, suggesting its binding to this protein.

FIG. 3.

Comparison of charges of different endonucleases. The structures of LEI, DNase I, and CAD were analyzed with Protein Explorer (23). Perpendicular views of cleaved LEI (PDB number 1HLE) (top), DNase I (PDB number 3DNI) (middle), and CAD (PDB number 1V0D) (bottom) were charge colored (basic regions are blue, and acidic regions are red). DNA binding regions of the three molecules are more basic than the opposite side. That area would easily interact with acidic charges of DNA. Aspartate and histidine residues that seem important for endonuclease activity are yellow. DNA associated with DNase I is green. CAD seems to be polarized slightly differently, maybe because it is the only molecule that works as a dimer.

FIG. 4.

Site-directed mutagenesis of the consensus bipartite NLS. (a) Impairment of nuclear translocation is shown for the K225A NLS LEI mutant. Cotransfection was done with a green fluorescent protein-LEI plasmid to validate the nuclear translocation of wild-type LEI. The right part of the panel shows the different distributions of wild-type and NLS/K225A mutant LEI. Pictures were taken 16 h after induction of apoptosis with 40 μM EIPA. Nuclei or apoptotic bodies containing nuclear material are indicated by broken outlines. (b) The lysines of the putative L-DNase II NLS were changed to alanines, and it was cotransfected with a wild-type LEI construct into BHK cells (as described in Materials and Methods). Evaluation of the nuclearization of the constructs during apoptosis induced with 40 μM EIPA is summarized on the left. Each number on the left corresponds to the last number of the mutated lysine. Green squares represent wild-type behavior of the mutated protein, and red squares represent impaired translocation. Black and gray squares represent no or little fluorescent proteins, and hatched squares indicate the presence of fluorescent aggregates in cells. (c) Impairment of the nuclear translocation of a mutated lysine was scored 1. This allowed the assignment of a score to each mutant represented on the histogram. Lysine 225 is the most important for L-DNase II nuclearization. (d) The K225A LEI mutant is impaired in the ability to fix importin α compared to wild-type (wt) LEI. Calmodulin was used as a negative pull-down control, as it does not bind directly to importin α, and a brut extract of HeLa cells was used as a positive control for the presence and molecular mass of importin α. (e) Proapoptotic activity of wild-type LEI. The rate of survival of BHK cells overexpressing different LEI constructs shows increased cell death for wild-type LEI, compared to the mutated constructs. Vectors: empty (yellow), LacZ (dark blue), wild-type LEI (green), H368A/endonuclease active site LEI mutant (pink), K225A/NLS LEI mutant (light blue), and nontransfected cells (red). Data are means ± standards deviations. *, P < 0.001 (Student's test) versus H368A and K225A. (f) Endonuclease activity of the K225A NLS LEI mutant. Site-directed mutagenesis was performed on the middle lysine of the major cluster of lysines of the NLS. The K225A LEI mutant was then incubated with elastase overnight at 37°C to obtain the cleaved form of LEI. Incubation of this cleaved form with plasmid DNA for 10 to 30 min allowed us to measure the endonuclease activity of the K225A LEI mutant and compare it with that of wild-type (wt) LEI. K225A LEI showed endonuclease activity comparable to that displayed by wild-type LEI previously treated with elastase. NLS mutant or wild-type LEI was not able to cleave DNA if it had not been treated previously with elastase. Plasmid DNA showed no autodegradation or cleavage by elastase.

FIG. 5.

Molecular structure of LEI and its implication in cellular fate. (a) The structure of LEI was analyzed with Protein Explorer. Native LEI (upper part) shows the RCL (green) covering the major cluster of lysines of the bipartite NLS (red). The endonuclease active site is orange. This three-dimensional structure was obtained from the pig LEI sequence. The lower part of the panel shows cleaved horse LEI (PDB number 1HLE) with an unmasked NLS (red) after insertion of the RCL into β-sheet A of serpin. (b) In unstressed cells, LEI is localized in the cytoplasm and has an antiprotease activity that preferentially inhibits elastase. When the cell undergoes a stress that causes the transformation of LEI into L-DNase II, the antiprotease activity is released. L-DNase II enters the nucleus, cleaves the DNA, and finally leads to apoptosis. Different mutant forms of LEI have been constructed to understand the LEI/L-DNase II pathway. NLS mutant LEI (K225A, red) is not able to enter the nucleus, and endonuclease active-site mutant LEI (H368A, green) is able to enter the nucleus but is unable to degrade DNA. In both cases, apoptosis is reduced. LEI has to be cleaved into L-DNase II and translocated to the nucleus to induce apoptosis.