R OLE OF CALCINEURIN THIOLS IN REDOX REGULATION OF THE PHOSPHATASE ACTIVITY

The discovery of calcineurin inhibition by PAO and MEL provided the basis to suggest that calcineurin activity can be subject to redox control through the oxidation/reduction of protein cysteines. Furthermore, the chemistry of PAO allows the hypothesis that two closely spaced cysteines on the enzyme molecule are the mechanism for calcineurin redox sensitivity. PAO and MEL form stable cyclic dithioester products with protein vicinal dithiols, which can be specifically reduced by low molecular weight dithiol compounds. Indeed, this was the case for the reaction of arsonous acid derivatives with calcineurin, since almost complete reactivation was achieved after incubation of the PAO- or MEL-inhibited enzyme with DTT or DMPS.

Arsenic-bridged protein derivatives resemble an intramolecular disulfide bridge. This indicated that calcineurin activity could be regulated via dithiol-to-disulfide transitions. The experiments with H2O2 inactivation of isolated bovine calcineurin provided evidence in support of this hypothesis. First, the inactivation by H2O2 could be significantly reversed by DTT and, more importantly, by thioredoxin, an enzyme specific for the reduction of disulfide bridges on proteins. Second, the analysis of free thiol groups revealed the elimination of 2-3 free cysteines after calcineurin treatment with H2O2. Taken together, these results show that thiol oxidation, most likely through disulfide bridge formation, inhibits calcineurin activity.

An important finding of the present study is the ability of thioredoxin to reactivate oxidized calcineurin. Thioredoxin/thioredoxin reductase system plays a critical role in repair of oxidative damage to the cell by reducing disulfide bonds in many proteins [Holmgren, 1985]. The fact that thioredoxin easily restored the activity of calcineurin after H2O2 treatment suggests possible significance of such oxidation-reduction processes in vivo.

The possibility of calcineurin regulation through dithiol-disulfide equilibrium was also illuminated in several other recent studies. Thus, depletion of thiols in the incubation medium of human NK-cells resulted in significant decrease of calcineurin activity in cellular extracts together with inhibition of calcineurin-to-NFAT signaling [Furuke et al., 1999]. In another study, activity of calcineurin in Jurkat cell lysates after inactivation by H2O2 was partly restored by treatment with DTT [Reiter et al., 1999]. These results, together with earlier data of calcineurin sensitivity to thiol-modifying reagents [King, 1986], support the role of calcineurin thiols in regulating the enzyme phosphatase activity.

The major question arising after discovery of involvement of calcineurin cysteines in regulation of enzyme activity was: which cysteines participate in such a regulation. The published X-ray structure of bovine calcineurin provided a basis for an indirect assessment.

Bovine calcineurin A and B subunits have 12 and 2 cysteines respectively. No disulfide bridges are found in the native form of calcineurin. The recombinant Dictyostelium discoideum calcineurin A subunit exhibited the same behavior towards PAO as bovine calcineurin, excluding the B subunit as a possible target. Furthermore, alignment of primary sequences of bovine and Dictyostelium discoideum catalytic subunits revealed that only nine cysteines are conserved between two species. The calculation of all distances between each possible pair among these nine cysteines identified the pair Cys228/Cys256 in bovine enzyme as the most likely candidate, having inter-thiol distance of 4.6Å. Inspection of the X-ray structure of bovine calcineurin revealed that Cys228 and Cys256 are located close to the substrate-binding cleft and also near to the binding site of the autoinhibitory domain (Fig.4.1.).

Figure 4.1. Positions of Cys228 and Cys256 in X-ray structure of bovine calcineurin. The cysteine residues are highlighted in yellow. The figure was created using public domain software Cn3D (http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml).

However, neither of these cysteines nor any other cysteines are close to the active site binuclear metal center, indicating that a direct interaction with the active site is unlikely.

Cys256 is located near Arg254, the residue critical for the enzyme catalytic activity. Thus, dislocation of this residue following disulfide bridge formation might explain the effects of dithiol-disulfide transition on the enzyme activity. However, direct data showing the involvement of Cys228/Cys256 in the regulation of calcineurin phosphatase activity were lacking.

To investigate the involvement of Cys228/Cys256 in redox regulation of calcineurin we resorted to the strategy of site-directed mutagenesis of these residues, exchanging them for redox-neutral alanines. Since recombinant Dictyostelium discoideum calcineurin A subunit was easily available through a collaboration with the group of Rupert Mutzel, Universität Konstanz, and it underwent the same inactivation after treatment with PAO, it was decided to mutate the homologous cysteines on Dictyostelium discoideum protein, namely, Cys278 and Cys305, either alone or in combination. Corresponding single and double mutations were introduced into plasmid DNA encoding N-terminally truncated, 6×His-tagged Dictyostelium discoideum calcineurin A, and the recombinant proteins were overexpressed and isolated on a Ni-NTA affinity column according to the standard procedures.

The recombinant Dictyostelium discoideum calcineurin A exhibited relatively low activity towards pNPP even in the presence of saturating amounts of divalent metal activators.

The attempts to further increase the enzyme activity by adding Dictyostelium discoideum calcineurin B subunit were not successful, indicating possible misfolding of the overexpressed protein. When effects of PAO and H2O2 on the activity of recombinant proteins were tested, no significant difference in sensitivity towards either of the reagents were observed between wild-type and mutated proteins. The sensitivity of Dictyostelium discoideum calcineurin A towards PAO and H2O2 was comparable with that of the bovine enzyme. However, it should be noted that recombinant calcineurin catalytic subunit seemed to be less stable towards oxidation compared to the bovine enzyme, since it was rapidly inactivated when TCEP was absent from the assay buffer. Again, this might be explained by partial misfolding of the protein and/or the exposure of cysteine residues otherwise masked by the interaction with the regulatory subunit. The calcineurin B-binding domain of the catalytic subunit constitutes a long amphipathic helix, which in the absence of B subunit might be destabilized. One cysteine residue, Cys372 is present at the end of this helix, and it could contribute to redox-sensitivity of isolated catalytic subunit of calcineurin.

In a recent study, Rusnak and associates created several other cysteine-to-alanine mutants of rat calcineurin A [Reiter et al., 1999]. We had the possibility to test the redox-sensitivity of the corresponding recombinant proteins, i.e. C88A/C197A, C197A, and C166A.

Again, these mutations did not lead to changes in calcineurin sensitivity towards PAO.

Therefore, the question, which cysteine(s) confer calcineurin sensitivity towards arsenic compounds and H2O2, and by which mechanism such an inhibition might occur, remains open. Several considerations arise from the results of the present studies. First, the inhibition through cysteine modification should not necessarily involve the residues in the immediate vicinity of the enzyme active center. Disulfide bond formation may be a consequence of relatively large changes of protein conformation, involving movements of entire protein domains. This could be particularly critical for calcineurin, which has a highly modular structure, and is regulated in a complex manner through a series of conformational changes after binding of calcium-loaded calmodulin. The exact nature of these changes is poorly investigated, and more structural data are needed to assess the movements of protein domains, and protein cysteines, upon calcineurin activation. The fact that PAO changes the enzyme sensitivity to calmodulin supports the hypothesis that it could interfere with conformational changes induced by calmodulin binding.

One should also consider that H2O2-induced oxidation can be rather non-specific, since it takes place at relatively high peroxide concentrations. Partial dimerization of calcineurin after H2O2 treatment indicates that surface-exposed cysteine residues might also be involved in the mechanisms of calcineurin oxidative inactivation. Such dimerization could, for example, hinder the substrate access to the catalytic center or prevent conformational changes leading to the enzyme activation. Thus, cysteines on calcineurin surface should not be excluded as possible targets of redox regulation of calcineurin phosphatase activity.

Additionally, the specificity of PAO for closely spaced cysteines should not be taken for granted. Although the inhibition was specifically relieved by dithiol-containing reagents, this can reflect the stability of the resulting product, but not the nature of the protein-PAO adduct. Therefore, it is possible that only a single cysteine is involved in the inhibition of calcineurin by PAO.

4.2. SUPEROXIDE INHIBITION OF CALCINEURIN BY TARGETING ITS FE²⁺-ZN²⁺ BINUCLEAR