Gangadhariah Mahesha H, Wang Benlian, Linetsky Mikhail, Henning Christian, Spanneberg Robert, Glomb Marcus A, Nagaraj Ram H
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH 44106, USA.
Biochim Biophys Acta. 2010 Apr;1802(4):432-41. doi: 10.1016/j.bbadis.2010.01.010. Epub 2010 Jan 18.
AlphaA-crystallin is a molecular chaperone; it prevents aggregation of denaturing proteins. We have previously demonstrated that upon modification by a metabolic alpha-dicarbonyl compound, methylglyoxal (MGO), alphaA-crystallin becomes a better chaperone. AlphaA-crystallin also assists in refolding of denatured proteins. Here, we have investigated the effect of mild modification of alphaA-crystallin by MGO (with 20-500 microM) 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 beta(L)- and gamma-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 beta(L)- and gamma-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 alphaA-crystallin to assist in the refolding of denatured MDH. However, MGO modification of chaperone-null TNS-bound alphaA-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 alphaA-crystallin but such modifications do not change its ability to assist in refolding of denatured proteins, 2) the sites on the alphaA-crystallin responsible for the chaperone function and refolding are the same in the native alphaA-crystallin and 3) additional hydrophobic sites exposed upon MGO modification, which are responsible for the enhanced chaperone function, do not enhance alphaA-crystallin's ability to refold denatured proteins.
αA-晶状体蛋白是一种分子伴侣；它可防止变性蛋白质聚集。我们之前已经证明，经代谢性α-二羰基化合物甲基乙二醛（MGO）修饰后，αA-晶状体蛋白会成为更好的伴侣蛋白。αA-晶状体蛋白还有助于变性蛋白质的重折叠。在此，我们研究了MGO（浓度为20 - 500微摩尔）对αA-晶状体蛋白的轻度修饰对其伴侣功能及其重折叠变性蛋白质能力的影响。在所使用的条件下，轻度修饰的蛋白质主要含有氢化咪唑酮修饰。修饰后的蛋白质对β(L)-和γ-晶状体蛋白、柠檬酸合酶（CS）、苹果酸脱氢酶（MDH）和乳酸脱氢酶（LDH）的热聚集以及胰岛素的化学聚集的伴侣功能增强。MGO轻度修饰后，该蛋白质协助化学变性的β(L)-和γ-晶状体蛋白、MDH和LDH重折叠以及防止CS热失活的能力未改变。催化无活性的热变性MDH或疏水探针2-对甲苯氨基萘-6-磺酸盐（TNS）预先结合会消除αA-晶状体蛋白协助变性MDH重折叠的能力。然而，伴侣蛋白缺失的TNS结合的αA-晶状体蛋白经MGO修饰后，伴侣功能部分恢复。综上所述，这些结果表明：1）氢化咪唑酮修饰足以增强αA-晶状体蛋白的伴侣功能，但此类修饰不会改变其协助变性蛋白质重折叠的能力；2）αA-晶状体蛋白上负责伴侣功能和重折叠的位点在天然αA-晶状体蛋白中是相同的；3）MGO修饰后暴露的额外疏水位点负责增强的伴侣功能，但不会增强αA-晶状体蛋白重折叠变性蛋白质的能力。
