br Molecular characterization of LEI L DNase II
Molecular characterization of LEI/L-DNase II LEI, like most serpins, is a metastable protein and its anti-protease activity is related to its “stressed to relaxed transition”. So that, the inhibition of elastase by the well described suicide mechanism generates the cleaved form of LEI (see Padron-Barthe et al. and Huntington et al. ,  for the detailed description of the mechanism). Interestingly, the analysis of the three-dimensional crystal structure of cleaved equine LEI showed a quite polarized molecule with a higher number of positive charges in the RSL pole. This is a necessary condition to interact with DNA, which is negatively charged. (Fig. 1). Although we could not reveal any consensus sequence for endonuclease activity, analysis of LEI three-dimensional structure showed the presence of two histidines (H287 and H368) in the more positively charged region . From these histidines, we focused on H368 as a candidate for being important at the active site because it is conserved in all species. The introduction of a point mutation on this residue (H368A) results in a molecule that conserves its anti-protease activity but has lost its endonuclease activity. These results suggest that the insertion of the RSL in the main beta-sheet uncovers a pre-existing endonuclease active site, related to H368. Moreover, our site-directed mutagenesis studies also showed that the RSL also hides a bipartite nuclear localization signal (NLS) which becomes exposed at the same time that the endonuclease active site, allowing then the nuclear translocation of the cleaved molecule. A molecular model of the shift in activity and cellular localization of LEI is seen on Fig. 2. The containment of DNA in the cell nucleus permits the separation of the genomic information from processes located in the cytoplasm. This is important to regulate the activity of some proteins. The general means of controlling the proteins shuttle between nucleus and PX 866 is through the presence of an NLS (allowing the protein to be introduced in the nucleus) and, in some proteins, of a Nuclear Extrusion Signal (NES). The NES consists on a motif containing three or four hydrophobic residues that bind a soluble export carrier: Crm1/exportin1. We have found a consensus NES sequence in LEI that binds Crm1 and ensures the nuclear export of the molecule . Taken together these results allowed us to conclude that LEI/SERPINB1 intrinsically possesses two enzymatic activities: an anti-protease activity dependent on its reactive site loop, which is analogous to the other proteins of the family and an endonuclease activity which is unveiled by the cleavage of the RSL by the cognate protease after the conformational modification induced by this cleavage, thought the same mechanism of inhibition of all serpins . In addition the conformational change also unveils a bipartite NLS allowing the protein to translocate to the nucleus. LEI displays also a functional NES sequence allowing to control, by nuclear exclusion of the DNase activity of L-DNase II.
LEI/L-DNase II in apoptosis The orderly cell dismantling seen in apoptosis involves proteolysis and chromatin degradation. The degradation of DNA in oligonucleosomes (DNA laddering) was considered in the earliest 90′ as the molecular hallmark of apoptosis. The search of DNases became then an important goal in apoptosis research . Many enzymes were proposed as candidates, but most did not fulfill the criteria for the apoptotic DNase. At that time, many results indicated the existence of different apoptotic pathways but these results were neglected due to the increasing knowledge on caspases activation and the discover of CAD (Caspase-Activated DNase). Since then it has been shown that the apoptotic phenotype of a dying cell can be obtained by the activation of other proteases, including calpains, cathepsins and granzymes . These proteases may activate, directly or indirectly, other endonucleases than CAD. To date, only the activation of: GAAD (Granzyme A-Activated DNase) and L-DNase II have been related to the activation of proteases , . Of note, the release of AIF (Apoptosis Inducing Factor) has also been related to protease activities (calpains) although its action on DNA is not to cleave it but to condense chromatin .