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Reactive cysteine in the structural Zn(2+) site of the C1B domain from PKC?.

ABSTRACT: Structural cysteine-rich Zn(2+) sites that stabilize protein folds are considered to be unreactive. In this article, we identified a reactive cysteine residue, Cys151, in a treble-clef zinc finger with a Cys(3)His coordination sphere. The protein in question is the C1B domain of Protein Kinase C? (PKC?). Mass-tagging cysteine assays of several C1B variants were employed to ascertain the site specificity of the covalent modification. The reactivity of Cys151 in C1B also manifests itself in the structural dynamics of the Zn(2+) coordination sphere where the S? of Cys151 alternates between the Zn(2+)-bound thiolate and free thiol states. We used NMR-detected pH titrations, ZZ-exchange spectroscopy, and residual dipolar coupling (RDC)-driven structure refinement to characterize the two exchanging conformations of C1B that differ in zinc coordination. Our data suggest that Cys151 serves as an entry point for the reactive oxygen species that activate PKC? in a process involving Zn(2+) release.

SUBMITTER: Stewart MD

PROVIDER: S-EPMC3645944 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Reactive cysteine in the structural Zn(2+) site of the C1B domain from PKCα.

Stewart Mikaela D MD Igumenova Tatyana I TI

Biochemistry 20120905 37

Structural cysteine-rich Zn(2+) sites that stabilize protein folds are considered to be unreactive. In this article, we identified a reactive cysteine residue, Cys151, in a treble-clef zinc finger with a Cys(3)His coordination sphere. The protein in question is the C1B domain of Protein Kinase Cα (PKCα). Mass-tagging cysteine assays of several C1B variants were employed to ascertain the site specificity of the covalent modification. The reactivity of Cys151 in C1B also manifests itself in the st ...[more]

PMID: 22913772

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Project description:Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259-269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration-dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intra-molecular active site cysteine-lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Alzheimer's disease.

Dataset Information

Reactive cysteine in the structural Zn(2+) site of the C1B domain from PKC?.

Publications

Reactive cysteine in the structural Zn(2+) site of the C1B domain from PKCα.

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