Alphab-crystallin-assisted reactivation of glucose-6-phosphate dehydrogenase upon refolding.
Alphab-crystallin, a small heat-shock protein has been shown to prevent the aggregation of other proteins under various stress conditions. We have investigated the role of alphaB-crystallin in the reactivation of denaturant [GdmCl (guanidinium chloride)]-inactivated G6PD (glucose-6-phosphate dehydrogenase). Studies indicate that unfolding and inactivation of G6PD by GdmCl proceeds via formation of a molten globule-like state at low concentrations of GdmCl, which was characterized by having maximum surface hydrophobicity and no catalytic activity. At high concentrations of GdmCl, G6PD was completely unfolded, which upon dilution-induced refolding yielding 35% of original activity. In contrast, no activity was recovered when G6PD was refolded from a molten globule-like state. Interestingly, refolding of completely unfolded G6PD in the presence of alphaB-crystallin resulted in 70% gain of the original activity, indicating that alphaB-crystallin assisted in enhanced refolding of G6PD. Intriguingly, alphaB-crystallin was unable to reactivate G6PD from a molten globule-like state. Size-exclusion chromatography data indicate that alphaB-crystallin-assisted reactivation of completely unfolded G6PD is concomitant with the restoration of the native structure of G6PD. Nonetheless, alphaB-crystallin failed to reactivate G6PD from preformed aggregates. Moreover, methylglyoxal-modified alpha-crystallin, which occurs in aged and diabetic cataract lenses, was less efficient in the reactivation of denaturant inactivated G6PD. Diminished chaperone-like activity of alpha-crystallin due to post-translational modifications may thus result in the accumulation of aggregated/inactivated proteins.
Kumar
M Satish
MS
National Institute of Nutrition (ICMR), Jamai Osmania, Hyderabad 500 007, India.
Reddy
P Yadagiri
PY
Sreedhar
B
B
Reddy
G Bhanuprakash
GB
D000818:Animals; D002417:Cattle; D004793:Enzyme Reactivators; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000378:metabolism*; D020673:Protein Renaturation; D038203:alpha-Crystallin B Chain; D038201:alpha-Crystallins
C059215:CRYAB protein, human; D004793:Enzyme Reactivators; D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; D038203:alpha-Crystallin B Chain; D038201:alpha-Crystallins; D005954:Glucosephosphate Dehydrogenase; D011494:Protein Kinases; D019791:Guanidine