Hydroimidazolone modification of human αA-crystallin: Effect on the chaperone function and protein refolding ability

Abstract

Alpha A-crystallin is a molecular chaperone; it prevents aggregation of denaturing proteins. We have previously demonstrated that upon modification by a metabolic α-dicarbonyl compound, methylglyoxal (MGO), αA-crystallin becomes a better chaperone. Alpha A-crystallin also assists in refolding of denatured proteins. Here, we have investigated the effect of mild modification of αA-crystallin by MGO (with 20-500 μM) on the chaperone function and its ability to refold denatured proteins. Under the conditions used, mildly modified protein contained mostly hydroimidazolone modifications. The modified protein exhibited an increase in chaperone function against thermal aggregation of βL- and γ-crystallins, citrate synthase (CS), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and chemical aggregation of insulin. The ability of the protein to assist in refolding of chemically denatured βL- and γ-crystallins, MDH and LDH, and to prevent thermal inactivation of CS were unchanged after mild modification by MGO. Prior binding of catalytically inactive, thermally denatured MDH or the hydrophobic probe, 2-p-toluidonaphthalene-6-sulfonate (TNS) abolished the ability of αA-crystallin to assist in the refolding of denatured MDH. However, MGO-modification of chaperone-null TNS-bound αA-crystallin resulted in partial regain of the chaperone function. Taken together, these results demonstrate that: 1) hydroimidazolone modifications are sufficient to enhance the chaperone function of αA-crystallin but such modifications do not change its ability to assist in refolding of denatured proteins, 2) the sites on the αA-crystallin responsible for the chaperone function and refolding are the same in the native αA-crystallin and 3) additional hydrophobic sites exposed upon MGO modification, which are responsible for the enhanced chaperone function, do not enhance αA-crystallin's ability to refold denatured proteins.

Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

“…The chaperone assays were carried out as previously described [36]. The following ratios of αA-crystallin to client proteins (w/w) were used: αA-crystallin:citrate synthase-1:14; αA-crystallin:βL-crystallin- 1:30; αA-crystallin:γ-crystallin- 1:12; αA-crystallin:lysozyme-1:4.…”

Glyoxalase 1 Modulation in Obesity and Diabetes

“…This occurs as a consequence of the use of high energy triosephosphate intermediates in glycolytic metabolism (136). MG modification of alpha-crystallin improved chaperone function and protein refolding activity but this effect was limited to higher extents of protein modification by MG than found physiologically (59). However, accumulation of MG is implicated in host defence mechanisms -particularly anti-microbial activity of phagocytic cells.…”

Exploring Post-translational Arginine Modification Using Chemically Synthesized Methylglyoxal Hydroimidazolones

“…7 Notably, elevated levels of MGO and MG-H-adducts have been shown to induce a range of cellular and subcellular effects including perturbations in cell signaling, inhibition of protein synthesis and cell growth, 8 induction of oxidative stress and pro-inflammatory cytokine release, 9–14 decreases in the adhesive properties of vascular basement membrane, 15 alterations in chaperone function, 16 and numerous other processes. 17,18 MGO concentrations have also been observed to increase in various disease states, including diabetes, 19–22 cancer, 23 cardiovascular disease, 24 and renal failure.…”