The Influence of Some Post-Translational Modifications on the Chaperone-Like Activity of α-Crystallin

Boekel, M.A.M. van; Hoogakker, S.; Harding, John J.; Jong, Wilfried W. de

doi:10.1159/000267940

Cited by 69 publications

(50 citation statements)

References 4 publications

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“…Future investigations will be needed to determine how autophosphorylation of ␣B-crystallin is involved in the chaperone functions of ␣B-crystallin during unfolding and refolding reactions. In one report, the phosphorylated forms of ␣A-and ␣B-crystallins suppressed the unfolding and aggregation of ␤-crystallin at 60°C better than nonphosphorylated forms (41). In addition, ␣A-and ␣B-crystallins have been shown to prevent the cytochalasin-induced depolymerization of actin in a phosphorylation-dependent manner (42).…”

Section: Discussionmentioning

confidence: 99%

ATP-enhanced molecular chaperone functions of the small heat shock protein human αB crystallin

Muchowski

Clark

1998

Proc. Natl. Acad. Sci. U.S.A.

142

111

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We report direct experimental evidence that human ␣B-crystallin, a member of the small heat shock protein family, actively participates in the refolding of citrate synthase (CS) in vitro. In the presence of 3.5 mM ATP, CS reactivation by ␣B-crystallin was enhanced approximately twofold. Similarly, 3.5 mM ATP enhanced the chaperone activity of ␣B-crystallin on the unfolding and aggregation of CS at 45°C. Consistent with these findings, cell viability at 50°C was improved nearly five orders of magnitude in Escherichia coli expressing ␣B-crystallin. SDS͞PAGE analysis of cell lysates suggested that ␣B-crystallin protects cells against physiological stress in vivo by maintaining cytosolic proteins in their native and functional conformations. This report confirms the action of ␣B-crystallin as a molecular chaperone both in vitro and in vivo and describes the enhancement of ␣B-crystallin chaperone functions by ATP.Molecular chaperones of the large heat shock protein families not only suppress protein unfolding and aggregation in response to stress but also actively participate in the refolding of denatured proteins in vitro, often in an ATP-dependent manner (1, 2). ␣B-crystallin is a protein that shares sequence and functional similarities with small heat shock proteins (sHsps) from numerous species (3, 4). ␣B-crystallin is expressed constitutively at high levels in the lens of the eye and at lower levels in many nonlens cells and tissues and is up-regulated dramatically in response to stress and pathological conditions in vivo (3, 5). Chaperone-like activity has been described for ␣B-crystallin from humans and various other species in suppressing protein unfolding and aggregation in response to thermal or chemical stress (4,(6)(7)(8)(9). Although the participation of ␣-crystallin and other sHsps in the refolding of proteins has been reported (4, 6, 10), the enhancement by ATP on the reactivation and refolding of proteins by ␣B-crystallin has not. To functionally characterize ␣B-crystallin as a molecular chaperone, its effects on the unfolding and refolding of citrate synthase (CS) in the presence and absence of ATP were studied in vitro. CS was chosen as a model protein for the characterization of unfolding and refolding reactions because it has been used extensively with molecular chaperones that include GroEL, Hsp90, and the sHsps (6,(11)(12)(13)(14).ATP is an abundant phosphorous metabolite in lenses from many species (15) that is present in lens cells at concentrations as high as 6.7 mM (16,17), which is among the highest levels of any cell in the body (18). High concentrations of ATP (4-8 mM) are found in skeletal muscle (19), in which high levels of ␣B-crystallin are expressed (20). The activity of GroEL and other chaperones on unfolding and refolding of proteins has been characterized at high concentrations of ATP (6,11,(21)(22)(23)(24), which may reflect a functional relationship between high concentrations of ATP and chaperone activity in all cells. The functional experiments with ␣B-crystallin in th...

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Section: Discussionmentioning

confidence: 99%

ATP-enhanced molecular chaperone functions of the small heat shock protein human αB crystallin

Muchowski

Clark

1998

Proc. Natl. Acad. Sci. U.S.A.

142

111

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“…The chaperone-like property of this crystallin has been investigated by several workers in order to gain insight in both the mechanism of this property and its relevance to the eye lens transparency (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). ␣-Crystallin from the old human lenses (28) and from the selenite-induced cataract lenses of an animal model (29) are found to exhibit decreased chaperone-like activity.…”

Section: From the Centre For Cellular And Molecular Biology Hyderabamentioning

confidence: 99%

Chaperone-like Activity and Temperature-induced Structural Changes of α-Crystallin

Raman

Rao

1997

Journal of Biological Chemistry

142

132

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␣-Crystallin is known to exhibit chaperone-like activity. We have studied its chaperone-like activity toward the aggregation of ␤ L -crystallin upon refolding of this protein from its unfolded state in guanidinium chloride. The chaperone-like activity of ␣-crystallin is less pronounced below 30°C and is enhanced above this temperature. The plot of percentage protection as a function of temperature shows two transitions; one at 30°C and another at around 55°C. We have performed steady state fluorescence, fluorescence polarization, fluorescence quenching, circular dichroism, sedimentation analysis, and gel filtration chromatography to probe the temperature-induced structural changes of ␣-crystallin. Our results show that at above 50°C, ␣-crystallin undergoes a transition to a multimeric molten globule-like state. Above 30°C, a minor but detectable perturbation in its tertiary structure occurs that might lead to the observed exposure of its hydrophobic surfaces. These results support our earlier hypothesis that ␣-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30°C involving enhanced or reorganized hydrophobic surfaces of ␣-crystallin is important for its chaperone-like activity. It is possible that a structural alteration induced by temperature forms a part of the general mechanism of chaperone function, because they are required to function more effectively at nonpermissible temperatures.␣-Crystallin, a multimeric protein composed of both acidic (␣ A ) and basic (␣ B ) subunits with the molecular mass of approximately 20 kDa, is a major protein of the eye lens. It is also expressed in other tissues such as heart, kidney, brain, muscles, etc. (1-4) and under certain diseased conditions (5-8). It shares both structural and sequence homology with small heat shock proteins and behaves in several ways like small heat shock proteins (9 -12). Its expression can be induced by thermal (9) or hypertonic (13) stress. It was believed to play only a structural role in the formation of the transparent and highly refractive tissue of the eye lens. However, the discovery of its presence in non-lenticular tissues and its homology with small heat shock proteins suggests that it might have other functional properties.␣-Crystallin is shown to prevent the heat-induced aggregation of other crystallins and enzymes like a molecular chaperone (14). The chaperone-like property of this crystallin has been investigated by several workers in order to gain insight in both the mechanism of this property and its relevance to the eye lens transparency (15-30). ␣-Crystallin from the old human lenses (28) and from the selenite-induced cataract lenses of an animal model (29) are found to exhibit decreased chaperone-like activity. The chaperone-like activity of ␣-crystallin might be important in the formation and maintenance of the eye lens transparency, and the loss of transparency can be attributed to the loss of function of ␣-crystallin (17).Earlier we inv...

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“…(C) Superose 6 elution profiles of a 0n2 m, or 4 mg ml −" sample of α-crystallin which has been denatured with 2 , 4 , 6 , and 7n5  guanidine hydrochloride. Peak numbers correspond to those indicated in chaperone-like activity for γ-crystallin almost identical to that of native α-crystallin (van Boekel et al, 1996 ;Doss et al, 1997). When α-crystallin is denatured by dialysis against the concentrations of guanidine hydrochloride used in experiments described herein, the resulting renatured protein sample is heat stable, but no longer possesses the ability to provide a chaperone-like protection for γ-crystallin.…”

Section: Denaturation\renaturation : Fplc and Sds-page Studiesmentioning

confidence: 94%

Studies of the Denaturation Patterns of Bovine Alpha-Crystallin Using an Ionic Denaturant, Guanidine Hydrochloride and a Non-Ionic Denaturant, Urea

Doss-Pepe

Carew

Koretz

1998

Experimental Eye Research

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The Influence of Some Post-Translational Modifications on the Chaperone-Like Activity of α-Crystallin

Cited by 69 publications

References 4 publications

ATP-enhanced molecular chaperone functions of the small heat shock protein human αB crystallin

ATP-enhanced molecular chaperone functions of the small heat shock protein human αB crystallin

Chaperone-like Activity and Temperature-induced Structural Changes of α-Crystallin

Studies of the Denaturation Patterns of Bovine Alpha-Crystallin Using an Ionic Denaturant, Guanidine Hydrochloride and a Non-Ionic Denaturant, Urea

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