I NHIBITION OF ISOLATED BOVINE CALCINEURIN BY OXIDANTS VIA DITHIOL - DISULFIDE

3.1.1. INHIBITION OF CALCINEURIN BY PAO. REVERSIBILITY BY DISULFIDE REDUCING AGENTS.

The identification of calcineurin as one of the major proteins binding to immobilized phenylarsine oxide (PAO) [Schmachtel, 1996] indicated that calcineurin might have reactive cysteine(s) specifically interacting with arsenic moiety of PAO. PAO and related trivalent arsenic compounds react with high affinity with closely spaced (vicinal) thiol groups of proteins, which are likely to form a disulfide bridge upon oxidation. To investigate if PAO affects calcineurin activity we incubated purified bovine calcineurin in the presence of different PAO concentrations at pH 8.05 and measured calcineurin activity using pNPP as a substrate and Mg²⁺ as activating cation. PAO caused a concentration-dependent inactivation of calcineurin as shown on Fig. 3.1.1.

Figure 3.1.1. Concentration dependence of calcineurin inhibition by PAO. Calcineurin activity towards pNPP was measured after 5 min incubation with indicated concentrations of PAO.

The IC50 for PAO inhibition is about 7-8 µM. Apparent activation observed at low PAO concentrations was due to an activating effect of the PAO diluent, DMSO, under experimental conditions. A similar inhibition was observed when calcineurin activity was measured at pH 7.4 using Mn²⁺ as activating metal ion. We also tested another arsonous acid derivative, melarsen oxide (MEL) [Happersberger et al., 1998], for its effects on calcineurin activity.

Similar to PAO, MEL inhibited calcineurin with IC50 ≈ 7-8 µM (data not shown). Noticeably, the inhibition occurred in the presence of 0.5 mM TCEP. Although TCEP is a good disulfide-reducing reagent [Getz et al., 1999], and it was used for calcineurin thiol protection in all subsequent studies, apparently it did not interfere with modification of calcineurin by PAO.

The time course of calcineurin inactivation by 50 µM PAO is shown on Fig. 3.1.2. The inactivation consisted of a rapid activity fall to ~50% of initial value at 2 min followed by a slower decrease to about 10% at 30 min. The control reaction without PAO showed no

significant activity decrease over incubation time. The inactivation did not follow first-order kinetics, but in general was consistent with an irreversible inactivation process.

Figure 3.1.2. Time course of bovine calcineurin inactivation by PAO. Calcineurin activity towards pNPP was measured continuously for 30 min after addition of 50 µM PAO into the assay at pH 8.05.

We also tested the effect of PAO on calmodulin-dependence of calcineurin activity.

Table 3.1.1. shows that before PAO addition calcineurin was 7-8-fold activated by calmodulin. After PAO treatment for 15 min calcineurin was only 2-2.5-fold activated by calmodulin. These data indicated that modification of calcineurin thiols by PAO might interfere with its activation by calmodulin.

Table 3.1.1. Calmodulin dependence of native and PAO-inhibited calmodulin. pNPP phosphatase activity of bovine calcineurin at pH 8.05 in the absence (-CaM) and in the presence (+CaM) of calmodulin was measured before and after treatment with 50 µM PAO for 15 min. The number in parenthesis indicates fold activation by calmodulin.

Activity, Units/mg

before PAO after PAO

-CaM 216±24 90±21

+CaM 1645±181 (7,6) 207±19 (2,3)

Modification of vicinal thiols by arsonous acids results in formation of cyclic dithioesters. Modified thiols can be regenerated from the PAO adduct by treatment with reagents containing vicinal thiol groups, such as DTT or 2,3-dimercapto-1-propane sulfonic acid (DMPS). In contrast, the reagents containing only a single thiol like cysteine or 2-mercaptoethanol do not reverse the modification [Joshi and Hughes, 1981]. Indeed, addition of 0.5 mM DMPS after calcineurin treatment with 40 µM PAO led to a time-dependent re-activation of calcineurin (Fig. 3.1.3). A similar rere-activation was achieved with 1 mM DTT.

However, 1 mM 2-mercaptoethanol did not significantly reactivate calcineurin after PAO

inhibition (data not shown). These data suggested that bridging of vicinal cysteine residues on calcineurin causes the inhibition.

Figure 3.1.3. Reactivation of bovine calcineurin after PAO treatment with DMPS. Calcineurin was inhibited by 40 µM PAO and its activity towards pNPP was measured after addition of 0.5 mM DMPS in a standard assay at pH 8.05.

3.1.2. INACTIVATION OF CALCINEURIN BY H2O2.

The sensitivity of calcineurin to thiol modifying reagents led us to investigate whether common oxidants affect its phosphatase activity. H2O2 is generally used in models of oxidative stress and its interaction with calcineurin was investigated. Calcineurin was pre-incubated with H2O2 at pH 8.05 followed by pNPP phosphatase activity measurements. As shown on Fig. 3.1.4A, upon incubation with 1 mM H2O2 calcineurin underwent time-dependent inactivation, which could be fitted to apparent first-order kinetics. Incubation without H2O2 and TCEP resulted in only slight inactivation of calcineurin (to about 80% of initial activity after 30 min at 30°C).

Figure 3.1.4. Inactivation of bovine calcineurin by H₂O₂. A. Calcineurin was pre-incubated with 1 mM H2O2 at pH 8.05 for indicated times and its pNPP phosphatase activity was determined. The curve is a fit to first-order inactivation giving apparent kin=0.053 min^-1. B. Replot of the apparent first-order inactivation constants obtained as above against H2O2 concentration.

The replot of the apparent first-order inactivation constant against H2O2 concentration gave a hyperbolic curve (Fig. 3.1.4B), indicating that the inactivation is not a simple bimolecular process. The inactivation of calcineurin by H2O2 occurred also at pH 7.4, but at a significantly slower rate (data not shown). We also tested whether calcineurin activity towards RII phosphopeptide substrate is inhibited by oxidants. The experiments using RII produced similar inhibition by PAO and H2O2 (data not shown), indicating that oxidative inactivation of calcineurin is not substrate-specific.

Calmodulin dependence of H2O2-oxidized calcineurin was also tested. In contrast to PAO, H2O2 did not change significantly the degree of calcineurin activation by calmodulin (data not shown).

Inorganic phosphate (Pi) is a competitive inhibitor of calcineurin, and in case of protein tyrosine phosphatases it could protect the enzyme active site against oxidation by H2O2 [Caselli et al., 1998]. However, addition of 10 mM Pi did not protect calcineurin against inactivation by 1 mM H2O2. Still, when added to the assay mixture, 10 mM Pi inhibited calcineurin activity to ~30% of control (data not shown). Thus, it seems unlikely that the phosphate-binding site of the calcineurin is the target of oxidative inactivation. The same lack of protection by Pi was also found in case of PAO inhibition.

3.1.3. ROLE OF THIOL OXIDATION IN H2O2-MEDIATED CALCINEURIN INACTIVATION.

PAO inhibition of calcineurin revealed the presence of reactive thiols necessary for calcineurin activity. To test whether protein thiols are involved in the calcineurin inhibition by H2O2, the inactivated protein was treated with thiol-reducing reagents and its pNPP phosphatase activity was tested. 15 min treatment with DTT (10 mM) restored calcineurin activity to ~75-80% of control (Fig. 3.1.5). Other thiol reductants, such as DMPS or TCEP, had similar effects (data not shown). We also tested two physiological disulfide-reducing substances, thioredoxin and GSH, for their re-activatory effects on H2O2-inhibited calcineurin. 10 µM thioredoxin could restore calcineurin activity to 70% of control (Fig.

3.1.5), whereas 10 mM GSH had only a marginal reactivating effect (data not shown). We also tested whether reconstitution of the complete thioredoxin system (thioredoxin plus thioredoxin reductase plus NADPH) could lead to improvement of thioredoxin reactivation.

No difference was observed between thioredoxin and thioredoxin/thioredoxin reductase.

Taken together, these results showed that physiological as well as chemical disulfide reducing agents can at least partly reverse calcineurin inactivation by H2O2.

To assess the effect of H2O2 on calcineurin cysteines directly the thiol content of calcineurin was measured spectrophotometrically using DTP. DTP has some advantages over the commonly used Ellman’s reagent, since DTP is more stable in solution at neutral pH and it reacts with protein thiols over broader pH range [Jocelyn, 1987].

Figure 3.1.5. Reactivation of H₂O₂-inactivated calcineurin. The protein was treated with 1 mM H2O2 for 30 min at 30°C and pH 8.05 and either left untreated or incubated for further 15 min with 10 µM thioredoxin (Trx) or 10 mM DTT.

Table 3.1.2. shows that H2O2 treatment resulted in the loss of 2-3 free –SH groups in calcineurin measured both under native or denaturing conditions. Thus, inactivation of calcineurin by H2O2 is accompanied by oxidation of the protein cysteines.

Table 3.1.2. Determination of free thiols in native and H₂O₂-treated calcineurin.

Calcineurin sample was treated 30 min with 1 mM H2O2 and the number of free thiols before (CaNred) and after treatment (CaNox) was determined using DTP as described. Number in parenthesis indicates the number of determinations.

CaNred CaNox ∆SH

Native 6.7±0.5 (4) 4.3±0.5 (5) 2.4

Denaturing 10.9±0.2 (2) 8.2±0.1 (2) 2.7

3.1.4. CALCINEURIN OXIDATION DOES NOT AFFECT THE PROTEIN SECONDARY STRUCTURE, BUT CAUSES PARTIAL DIMERIZATION.

To look for possible changes of protein secondary structure following calcineurin oxidation the circular dichroism (CD) spectra of the native and H2O2-treated enzymes were measured. The CD spectrum of the native calcineurin sample showed negative maxima at 208 nm and 222 nm, consistent with a protein with significant amount of α-helical structure (Fig.

3.1.6).

96kDa¾

66kDa¾

R Ox

Figure 3.1.6. CD spectrum of calcineurin. Native calcineurin sample is shown by the solid line and the H2O2-treated sample – by the dotted line.

No significant changes of the CD spectrum were observed in the H2O2-treated sample, indicating that the oxidation does not affect the secondary structure of calcineurin. α-helical content of 35% calculated from the spectrum by the method of Yang [Yang et al., 1986] was in agreement with a 34% α-helical content calculated from the X-ray structure of human calcineurin [Kissinger et al., 1995].

Changes of protein conformation or quaternary structure upon oxidation can be traced by protein behavior on SDS-PAGE under non-reducing conditions. Fig. 3.1.7. shows that after incubation with 1 mM H2O2 an additional band corresponding to the size of calcineurin dimer appeared on a gel. Therefore, oxidative inactivation of bovine calcineurin by H2O2 was accompanied by a partial dimerization of the protein.

Figure 3.1.7. Non-reducing SDS-PAGE of bovine calcineurin. Calcineurin samples (5 µg) before (R) and after (Ox) treatment with 1 mM H2O2 were separated by SDS-PAGE under non-reducing conditions and stained by Coomassie Blue.

3.2. SITE-DIRECTED MUTAGENESIS OF CYSTEINES IN DICTYOSTELIUM DISCOIDEUM CALCINEURIN.