Glutathione transferase-derived compounds as potential therapeutic agents

Abstract

Glutathione transferases are generally recognized for their role as antioxidant enzymes in phase II detoxification reactions. Recent studies identified a diverse range of other functions which are unrelated to detoxification. One such action is the specific inhibition of cardiac ryanodine receptor Ca{u00B2}{u207A} release channel. It is well documented that excessively active RyR2 channels are partly responsible for low store Ca{u00B2}{u207A} levels and defective Ca{u00B2}{u207A} release in heart failure. Therefore, inhibition of RyR2 is a potential strategy for the treatment of heart failure as it would help to maintain low RyR2 activity during diastole. Previous studies which examined the effects of truncating GSTM2 on its ability to alter Ca{u00B2}{u207A} release from SR and RyR2 channel activity identified the GSTM2 C terminal domain (GSTM2C) as the region responsible for the inhibitory effect. Although it supported the inhibition of RyR2, it did not support the activation of RyR1 channels which was shown by the full length protein. Activity of smaller fragments from the GSTM2C indicated that the helix 6 in the C terminal domain is critical for the inhibitory effect. Although fragments containing the helix 6 sequences inhibited Ca{u00B2}{u207A} release from cardiac SR and RyR2 channel activity, helix 6 sequence alone was not effective. Structural analysis on circular dichroism spectroscopy revealed the helix 6 sequence to be unstructured which may have explained the lack of activity, if the helical nature of the fragment was essential for its efficacy. On the other hand, the active fragment of GSTM2 responsible for the inhibition of RyR2 activity may involve not only helix 6 but also the flanking helices. Therefore, the main aims of the thesis were to identify the minimum fragment of GSTM2 which is capable of inhibiting RyR2, to determine the effect of mutations on the inhibitory effect, to confirm the region of RyR2 that interacts with GSTM2 and finally, to determine the effects of GSTM2 fragments on the contractility of ventricular cardiomyocytes. First, I examined the activity of RyR2 in the presence of two GSTM2C constructs with potentially destabilizing mutations in helix 6. The mutants, Fl57A and Y160A failed to inhibit cardiac RyR2 activity in single channel lipid biayer experiments and Ca{u00B2}{u207A} release from cardiac SR. Although they retained the helical structure as indicated in circular dichroism spectroscopy, tryptophan fluorescence indicated changes in folding. Interestingly, wild type GSTM2C inhibited cardiac RyR2 only at positive potentials, which may develop during Ca{u00B2}{u207A} efflux, but not at negative potentials. This further suggested that therapeutics mimicking the structure of GSTM2C may reduce excess Ca{u00B2}{u207A} release during diastole, which can lead to fatal arrhythmias. Previous experiments carried out in our laboratory also showed that H5678 fragment of GSTM2C (containing helices 5,6,7 and 8) is capable of binding to the DR3 region of RyR2 which explains the isoform specific activity of GSTM2. Tryptophan fluorescence quenching experiments that I performed confirmed the binding between H5678 and 22 amino acid fragment (1869-1890) of DR3 region, which further narrowed down the GSTM2 binding site in cardiac RyR2. Consistent with previous results, binding between the two proteins was weak with dissociation constants in micro molar range. Since DR3 is located in the clamp region of the 3D structure of RyR2, specific binding of GSTM2C to RyR2 could dramatically influence the gating of the channel. I investigated the effect of GSTM2C and the mutants, Fl57A and Yl60A on the contractility of neonatal cardiomyocytes. Consistent with the results obtained with single channel lipid bilyer experiments and Ca{u00B2}{u207A} release assays, the mutants failed to reduce the contractility of cardiomyocytes. Ability of GSTM2C to inhibit RyR2 activity hence Ca{u00B2}{u207A} release from SR was further indicated when the contractility of cardiomyocytes was reduced significantly in the presence of GSTM2C. A new property of GSTM2C was identified when the GSTM2C was shown to be capable of entering into cardiomyoctytes without a carrier cell penetrating peptide. The overall results presented in this study identify GSTM2C as a potential therapeutic for the treatment of heart failure. The helix 6 sequence in the GSTM2C and its flanking helices played a major role in stabilizing the molecule and in binding to the DR3 region of RyR2. Due to the voltage dependent activity, GSTM2C could be used during diastole to reduce the abnormal Ca{u00B2}{u207A} leak through ryanodine receptors. Thus, GSTM2C could be the only isoform specific, endogenous inhibitor of cardiac ryanodine receptor activity reported so far.