Glutathiolation enhances the degradation of gammaC-crystallin in lens and reticulocyte lysates, partially via the ubiquitin-proteasome pathway

Abstract

Purpose: S-glutathiolated proteins are formed in the lens during aging and cataractogenesis. The objective of this work was to explore the role of the ubiquitin-proteasome pathway in eliminating S-glutathiolated gammaC-crystallin.

Methods: Recombinant human gammaC-crystallin was mixed with various concentrations of glutathione (GSH) and diamide at 25 degrees C for 1 hour. The extent of glutathiolation of the gammaC-crystallin was determined by mass spectrometry. Native and S-glutathiolated gammaC-crystallins were labeled with (125)I, and proteolytic degradation was determined using both lens fiber lysate and reticulocyte lysate as sources of ubiquitinating and proteolytic enzymes. Far UV circular dichroism, tryptophan fluorescence intensity, and binding to the hydrophobic fluorescence probe 4,4'-dianilino-1,1'-binaphthalene-5,5'-disulfonic acid (Bis-ANS), were used to characterize the native and glutathiolated gammaC-crystallins.

Results: On average, two and five of the eight cysteines in gammaC-crystallin were glutathiolated when molar ratios of gammaC-crystallin-GSH-diamide were 1:2:5 and 1:10:25, respectively. Native gammaC-crystallin was resistant to degradation in both lens fiber lysate and reticulocyte lysate. However, glutathiolated gammaC-crystallin showed a significant increase in proteolytic degradation in both lens fiber and reticulocyte lysates. Proteolysis was stimulated by addition of adenosine triphosphate (ATP) and Ubc4 and was substantially inhibited by the proteasome inhibitor MG132 and a dominant negative form of ubiquitin, indicating that at least part of the proteolysis was mediated by the ubiquitin-proteasome pathway. Spectroscopic analyses of glutathiolated gammaC-crystallin revealed conformational changes and partial unfolding, which may provide a signal for the ubiquitin-dependent degradation.

Conclusions: The present data demonstrate that oxidative modification by glutathiolation can render lens proteins more susceptible to degradation by the ubiquitin-proteasome pathway. Together with previous results, these data support the concept that the ubiquitin-proteasome pathway serves as a general protein quality-control mechanism.

FIGURE 1

SDS-polyacrylamide gel electrophoresis of native and GSH-modified γC-crystallin. Samples were treated with Laemmli buffer, with or without β-mercaptoethanol as indicated, and resolved on 15% separating gels, and the gels were stained with Coomassie Blue. Native γC-crystallin is denoted native γC and glutathiolated γC-crystallins were referred to as γC-2GSH and γC-5GSH, depending on the number of GSH adducts per γC-crystallin molecule, as revealed by subsequent mass spectrometry (Fig. 2). Numbers on the left indicate molecular mass of the standards (kDa).

FIGURE 2

Mass spectrometry characterization of native and glutathiolated γC-crystallin. Native and glutathiolated γC-crystallins were analyzed with LC-MS, and the deconvoluted mass spectra are shown. (A) Native γC-crystallin; (B) γC-crystallin modified with GSH at a molar ratio of 1:2; (C) γC-crystallin modified with GSH at a molar ratio of 1:10. G0 represents unmodified γC-crystallin; G1 represents γC-crystallin modified with 1 glutathione; G2 represents γC-crystallin modified with two glutathiones; G3 represents γC-crystallin modified with three glutathiones; G5 represents γC-crystallin modified with five glutathiones; and G6 represents γC-crystallin modified with six glutathiones. *Adduct of γC-crystallin with single protease inhibitor (+ 182); **adduct of γC-crystallin with two protease inhibitors.

FIGURE 3

Proteolytic degradation of ¹²⁵I-labeled native and GSH-modified γC-crystallin. (A) Degradation assay performed using bovine lens fiber cell lysate, with or without addition of ATP. (B) Degradation assay performed using reticulocyte lysate as the source of UPP-components with (total degradation) and without addition of ATP/Ubc4. (C) ATP-/Ubc4-stimulated degradation in reticulocyte lysate. Experiments were performed in triplicate and repeated at least three times with two separate batches of γC-crystallin. Shown are pooled data from all degradation experiments: n ≥ 12. Mean ± SEM *P < 0.0005, †P < 0.025, #P < 0.05 (compared with native γC-crystallin with/without addition of ATP, respectively).

FIGURE 4

Involvement of the proteasome and ubiquitin in the degradation of glutathiolated γC-crystallin in reticulocyte lysate. (A) MG132 (final concentration 24 µM) or (B) HNE-Ub (HNE-ubiquitin, final concentration 10 µM) was added to reticulocyte lysate before the assay was started. Wild-type ubiquitin (WT-Ub) was used as the control. Data shown are the mean ± SEM of results in two independent experiments; each experiment was performed in triplicate. *P < 0.001.

FIGURE 5

Trp (A) and Bis-ANS (B) fluorescence spectra of native and GSH-modified γC-crystallin. The numbers refer to native γC-crystallin (1), γC-crystallin modified with an average of two glutathiones (2), and γC-crystallin modified with an average of five glutathiones (3). Protein concentrations of all substrates were 0.15 mg/mL in 50 mM Tris-HCl buffer, pH 7.6.

FIGURE 6

Far UV CD spectra of native and glutathiolated γC-crystallin. Far UV CD spectra for native γC-crystallin (1), γC-crystallin modified with an average of two glutathiones (2), and γC-crystallin modified with an average of five glutathiones (3) were determined. Protein concentrations were 0.2 mg/mL in 50 mM Tris-HCl buffer (pH 7.6). Cell path length was 1 mm.