Pier Giuseppe Vilardo

Specifically Targeted Modification of Human Aldose Reductase by Physiological Disulfides

Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303.

Oxidative modification of aldose reductase induced by copper ion. Definition of the metal-protein interaction mechanism.

Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys303 appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys298-Cys303. However, a Cys80-Cys303 disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys80-Cys303 disulfide to the more stable product Cys298-Cys303 is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys80-Cys303disulfide. However, no evidence of conditions favoring the formation of the Cys298-Cys303 disulfide was observed. Our proposal is that the generation of the Cys298-Cys303 disulfide, either directly or by rearrangement of the Cys80-Cys303 disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.

Modulation of aldose reductase activity through S-thiolation by physiological thiols.

The glutathionyl-modified aldose reductase (GS-ALR2) is unique, among different S-thiolated enzyme forms, in that it displays a lower specific activity than the native enzyme (ALR2). Specific interactions of the bound glutathionyl moiety (GS) with the ALR2 active site, were predicted by a low perturbative molecular modelling approach. The outcoming GS allocation, involving interactions with residues relevant for catalysis and substrate allocation, explains the rationale behind the observed differences in the activity between GS-ALR2 and other thiol-modified enzyme forms. The reversible S-glutathionylation of ALR2 observed in cultured intact bovine lens undergoing an oxidative/non oxidative treatment cycle is discussed in terms of the potential of ALR2/GS-ALR2 inter-conversion as a response to oxidative stress conditions.

Kinetics of human thrombin inhibition by two novel peptide inhibitors (Hirunorm IV and Hirunorm V).

A study on the kinetics of human thrombin inhibition by two novel synthetic peptides (Hirunorm IV and Hirunorm V) and a comparison with recombinant hirudin and a commonly used thrombin inhibitor, Hirulog-1, are reported. The dissociation constants for Hirunorm IV and Hirunorm V were determined by varying the concentration of inhibitors at fixed concentrations of the chromogenic substrate Chromozym-TH (N-tosylglycyl-L-prolyl-L-arginine 4-nitroanilide acetate). Both inhibitors behaved as reversible tight-binding inhibitors of amidolytic thrombin activity. The apparent dissociation constants determined showed a linear dependence on the concentration of substrate; this finding, which indicates that the inhibition was competitive, made possible the estimation of the dissociation constants (KI) for Hirunorm IV and Hirunorm V, which were 0.134 +/- 0.014 nM and 0.245 +/- 0.016 nM, respectively. Similar dissociation constants were also obtained for the two inhibitors when thrombin activity was measured with fibrinogen in the clotting assay. When tested for resistance to thrombin proteolytic activity, both inhibitors were inviolate to cleavage by thrombin. The data obtained demonstrate that both Hirunorm IV and Hirunorm V are potent and stable inhibitors of human thrombin activity.

Interconversion pathways of aldose reductase induced by thiol compounds.

The occurrence of protein S-thiolation as a consequence of oxidative stress is widely recognized (Thomas et al., 1990; Lou et at, 1990; Schuppe et al., 1992; Giblin, et al., 1995). However, the role and the relevance of this process are still a matter of debate.

Hirunorms, novel hirudin-like direct thrombin inhibitors

1. Hirunorms are new synthetic peptides designed to interact with thrombin in a similar way to the natural inhibitor hirudin. 2. Hirunorms are specific and efficient in vitro inhibitors of thrombin activity. 3. Hirunorms are potent anticoagulant and antithrombotic agents in in vivo experimental models devoid of hemorrhagic effects at doses that are active in preventing thrombosis.